KR102198971B1 - Novel Method for producing foot-and-mouth disease vaccine antigen - Google Patents

Abstract

The present invention relates to a novel method for preparing a foot-and-mouth disease vaccine antigen, and more specifically, by performing hydrophobic interaction chromatography on an inactivated foot-and-mouth disease virus to remove a non-structural protein (NSP) and obtain a foot-and-mouth disease vaccine antigen. It provides a method for preparing a foot-and-mouth disease virus vaccine antigen comprising the step of. In the case of removing non-structural proteins by flow-through method and easily preparing foot-and-mouth disease vaccine antigens through the hydrophobic interaction chromatography method of the present invention, limitations such as economical efficiency and reduction in vaccine antigen recovery rate occurring in the conventional method This was overcome. In addition, since there is no concern of induction of non-structural protein antibodies when inoculating the vaccine antigen according to the method of the present invention, it can be utilized as a safe and effective domestic foot-and-mouth disease vaccine antigen manufacturing technology.

Description

Novel method for producing foot-and-mouth disease vaccine antigen

The present invention relates to a novel method for preparing a foot-and-mouth disease vaccine antigen.

Foot-and-mouth disease (foot-and-mouth disease) is a viral infection in right-wing animals, causing symptoms such as high fever and blisters, and is the first domestic legal animal epidemic that causes death in young livestock. In Korea, it has occurred eight times since 2000, and in 2010-2011, it caused direct economic damage of about 3 trillion won.

Foot-and-mouth disease virus is composed of a total of four proteins (VP1, VP2, VP3, VP4). When the virus proliferates in a cell, the four proteins are gathered one by one to form a single body (protomer), and then five single bodies are gathered to form a pentamer. Thereafter, 12 pentamers are gathered to form a complete form of foot-and-mouth disease virus particles, and since the density and sedimentation coefficient of centrifugation are 146S, the complete particle form is sometimes referred to as 146S.

In addition, it has been reported that when the complete form of foot-and-mouth disease virus is decomposed into pentamers by high heat or low pH, the immunogenicity is much lower than that of the full form virus particles. Thus, foot-and-mouth disease in the step of preparing a foot-and-mouth disease vaccine or the finished product step It is a necessary situation to keep the virus particles intact.

Currently, the only way to control foot-and-mouth disease most effectively is to inoculate a preventive vaccine in advance to animals susceptible to foot-and-mouth disease. Domestically, from December 24, 2010 until now, foot-and-mouth disease vaccines are already being vaccinated nationwide in cattle, pigs, and goats.

In areas where foot-and-mouth disease vaccine is administered, the differential diagnosis between the vaccine-inoculated axis and foot-and-mouth disease infection axis is determined by the presence or absence of non-structural protein antibodies, so a process of removing non-structural proteins in the step of preparing the foot-and-mouth disease vaccine antigen is essential. However, since there is no known technology worldwide that can selectively remove only non-structural proteins with the current technology, a method of partially concentrating and purifying foot-and-mouth disease virus is used as the next best solution. Accordingly, in preparing foot-and-mouth disease vaccine antigens, various vaccine manufacturers usually precipitate foot-and-mouth disease virus using polyethylene glycol (polyethyleneglycol 6000 or 8000), store it in a concentrated antigen state at cryogenic temperature, and then dilute it to an appropriate multiple with a buffer solution. After that, it is mixed with a vaccine adjuvant and released as a final product.

However, when polyethylene glycol (PEG) is treated in this way, polymeric substances are usually precipitated, but some of the proteins of small molecular weight such as non-structural proteins are also mixed and precipitated.Therefore, there is a problem that the non-structural proteins of the foot-and-mouth disease virus are not completely removed. However, if the vaccine thus prepared is repeatedly inoculated to livestock several times, non-structural protein antibodies may appear, especially in cattle and goats. If the foot-and-mouth disease vaccine is repeatedly vaccinated in cattle and goats, as in Korea, there is a high possibility that the non-structural protein antibody of the foot-and-mouth disease virus is induced in livestock. According to the results of previous studies related to this, it is also known that non-structural protein antibodies are formed when the existing commercial vaccine is repeatedly inoculated more than 5 times.

Therefore, there is an urgent need for a method of producing a vaccine antigen at a level in which non-structural protein antibodies are not produced even when the non-structural protein is easily removed selectively and repeatedly inoculated more than 5 times as in Korea.

Accordingly, the present inventors have made extensive research efforts to develop a method for producing foot-and-mouth disease vaccine antigens in a state in which only non-structural proteins are selectively removed. As a result, the present inventors first constructed a chromatography method using a hydrophobic interaction column to separate foot-and-mouth disease virus and non-structural proteins based on the hydrophobic properties of non-structural proteins (especially, hydrophobic residues located at the N-terminus of 3AB). The present invention was completed by confirming that only complete particles of foot-and-mouth disease virus (146S) can be easily obtained in a state in which only non-structural proteins are bound to the column, thereby separating complete particles of foot-and-mouth disease virus and non-structural proteins.

Accordingly, an object of the present invention is to provide a method for easily preparing a foot-and-mouth disease vaccine antigen by removing only non-structural proteins using hydrophobic interaction chromatography.

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

According to one aspect of the present invention, the present invention provides a method for preparing a foot-and-mouth disease virus vaccine antigen comprising the following steps:

(a) inactivating foot-and-mouth disease virus; And

(b) removing the non-structural protein (NSP) by performing hydrophobic interaction chromatography on the inactivated foot-and-mouth disease virus in step (a) and obtaining a foot-and-mouth disease virus vaccine antigen.

According to a preferred embodiment of the present invention, the hydrophobic interaction chromatography in step (b) is a phosphate buffer solution containing 0.25 to 0.5M ammonium sulfate, and is hydrophobic under conditions of pH 7.5 to 8.0 and a flow rate of 1.0 to 2.0 ml/min. It is carried out by Fast Protein Liquid Chromatography using an interaction column.

According to a preferred embodiment of the present invention, the hydrophobic interaction chromatography in step (b) recovers a fraction not attached to the hydrophobic interaction chromatography column.

According to a preferred embodiment of the present invention, the foot-and-mouth disease virus vaccine antigen is obtained from the fraction that is not attached to the hydrophobic interaction chromatography column.

The foot-and-mouth disease virus supernatant non-binding elution method using a phosphate buffer solution containing ammonium sulfate of the present invention enables the recovery of complete foot-and-mouth disease virus particles from which non-structural proteins have been removed. The removal of non-structural proteins is a very important factor in the manufacturing stage of foot-and-mouth disease vaccine antigens and contributes to improving the quality competitiveness of the finished product.

That is, a feature of the present invention is that by separating foot-and-mouth disease virus particles and non-structural proteins using a hydrophobic interaction chromatography method, it is possible to obtain only foot-and-mouth disease virus particles, which are foot-and-mouth disease vaccine antigens, while selectively removing non-structural proteins easily. Have.

As long as the present invention can achieve the desired effect of separation of foot-and-mouth disease virus particles and non-structural proteins, (a) the method of inactivation of the step of inactivating foot-and-mouth disease virus is not limited, and any inactivation method known in the art Can also be used.

In one embodiment of the present invention, the hydrophobic interaction chromatography method was used by applying a hydrophobic interaction column (HiTrap Butyl Fast Flow Column, GE Healthcare) to AKTA Pure 150 (GE healthcare), but the object of the present invention It is not limited thereto, as long as it is possible to achieve separation of the paw-and-mouth disease virus particles and non-structural proteins, and any hydrophobic interaction chromatography method known in the art may be used.

In addition, as the column used in the chromatography, any column known in the art may be used as long as it can be used to remove non-structural proteins.

According to a preferred embodiment of the present invention, the foot-and-mouth disease virus vaccine antigen, the foot-and-mouth disease virus particle, is in the form of a complete particle (146S) or a form of a degraded particle (12S), more preferably in the form of a complete particle (146S).

In the present invention, the "foot-and-mouth disease virus particles" are referred to as 146S or 12S, respectively, depending on the sedimentation coefficient of centrifugation. The "complete particle" is a total of four proteins (VP1, VP2, VP3, VP4) of foot-and-mouth disease virus, one by one to form a single body (protomer), five single bodies are gathered to form a pentamer (pentamer), then pentamer It means 12 pieces.

According to a preferred embodiment of the present invention, the hydrophobic interaction in step (b) is a hydrophobic bond in which a hydrophobic residue of a hydrophobic interaction chromatography column ligand and a hydrophobic residue of a non-structural protein are bonded.

The hydrophobic residue may be one or more selected from the group consisting of butyl, phenyl, octyl, isopropyl, and ether.

In one embodiment of the present invention, the hydrophobic interaction column is a hydrophobic linking group that binds through a butyl ligand, but as long as the separation of the foot-and-mouth disease virus particles and the non-structural protein, which is the objective effect of the present invention, can be achieved, It is not limited, and a hydrophobic bond known in the art may be used.

According to a preferred embodiment of the present invention, the serotype of the foot-and-mouth disease virus is at least one serotype selected from the group consisting of O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1.

According to a preferred embodiment of the present invention, the foot-and-mouth disease virus is a domestic isolated foot-and-mouth disease type O virus.

In the present invention, the "foot-and-mouth disease serotype" has 7 serotypes, and the foot-and-mouth disease virus lacks 3D polymerase gene editing (3D pol gene proofreading) and post-copy repair activity in the replication process. Therefore, the serotype is present due to high antigenic variability.

In one embodiment of the present invention, only one column is used as a column using hydrophobic interaction, but the type and number of hydrophobic interaction columns are not limited in order to achieve the purpose of removing non-structural proteins.

More specifically, in the present invention, a hydrophobic interaction column (HiTrap Butyl Fast Flow Column, GE Healthcare) can be used, and various volumes are injected into the column through a 0.2 μm filter for the test sample, and the flow rate is 1 ml/min or more. It can be used at the flow rate allowed by the column. To bind to the hydrophobic ligand, a 20 mM phosphate buffer containing ammonium sulfate is used, and when eluting from the column after binding, only a 20 mM phosphate buffer containing no ammonium sulfate is used. Complete particles can be separated. Under the condition of 0.5 M ammonium sulfate, only non-structural proteins can be removed by binding to the column, and foot-and-mouth disease virus particles can be easily obtained from the flow-through fraction.

There is a similar case in which a vaccine antigen was prepared using a conventional hydrophobic interaction chromatography method, but the vaccine antigen recovery rate decreased when a domestic isolate was applied. In addition, the conventional method is a method of elution after binding foot-and-mouth disease virus to be purified to a hydrophobic interaction column, so the purification time is long and the cost is high. As such, the previously reported hydrophobic interaction column It is obvious that all of the applications are combined and eluted.

Therefore, in order to overcome this limitation, the present inventors first constructed a flow-through method, and as a result, solved the problem of lowering economic efficiency and vaccine antigen recovery rate. In particular, in the conventional method, it is not possible to determine whether the non-structural protein is removed, so there is a possibility of inadequateness in the vaccine purity evaluation according to the residual amount of the non-structural protein. It can be used as an effective domestic foot-and-mouth disease vaccine antigen manufacturing technology.

In addition, in the present invention, the foot-and-mouth disease virus supernatant is mixed with ammonium sulfate and injected into the column to bind only the non-structural protein to the ligand of the column, and the foot-and-mouth disease virus is recovered from the flow-through fraction, thereby saving time and cost and easily Conditions for producing foot-and-mouth disease vaccine antigens were established.

The most important success factor in the manufacturing process of foot-and-mouth disease vaccine is to effectively remove non-structural proteins and thereby minimize production costs. In the past, since there was no method to selectively remove only non-structural proteins, most foot-and-mouth disease vaccine manufacturers have mainly used ultrafiltration or PEG precipitation as a technique for partially concentrating and purifying the foot-and-mouth disease virus, but still the problem of remaining non-structural proteins has been resolved. There is a disadvantage in that non-structural protein antibodies are induced when repeated inoculations are performed 4-5 times. When the method presented in the present invention is applied, most of the non-structural proteins can be removed, and time and cost can be minimized, making it easy to obtain high purity (by evaluating whether to induce non-structural protein antibodies by repeatedly inoculating an animal with foot-and-mouth disease vaccine). It has the advantage of providing process conditions that can produce vaccine antigens.

In conclusion, the method of the present invention provides a high removal rate of non-structural proteins compared to the PEG method commonly used in the foot-and-mouth disease vaccine production process, and overcomes the limitations of the previously reported elution method after ligand binding of the virus. In terms of providing high efficiency. The technology for producing a foot-and-mouth disease vaccine antigen using the method of the present invention is the first in the world to be implemented by the present inventors, which can be very usefully utilized as a localization technology for foot-and-mouth disease vaccine.

In the case of preparing a foot-and-mouth disease vaccine antigen by a flow-through method through the hydrophobic interaction chromatography method of the present invention, limitations such as economic efficiency and reduction in vaccine antigen recovery rate occurring in the conventional method have been overcome. In addition, the foot-and-mouth disease vaccine antigen prepared by the method of the present invention can be used as a safe and effective domestic foot-and-mouth disease vaccine antigen manufacturing technology because non-structural proteins are removed, as well as non-structural protein antibodies are not detected during inoculation of the target animal. Facilitate monitoring. Therefore, it can contribute to the national defense system for foot-and-mouth disease and improve national competitiveness of foot-and-mouth disease vaccine.

1A is a chromatogram showing a series of processes in which ammonium sulfate is mixed in the supernatant of a foot-and-mouth disease type O Jincheonju virus, a domestic isolate, and then injected into a hydrophobic interaction column for liquid chromatography, and then eluted with a buffer solution containing no ammonium sulfate. The peak-1 and peak-2 fractions were collected and analyzed.
FIG. 1b shows the fractions corresponding to two peaks (peak-1: unbound eluted fraction, peak-2: eluted fraction after binding) obtained in chromatography were concentrated, respectively, and applied to the sucrose density gradient ultra-fast centrifugation method. As a result of measuring absorbance continuously by connecting a continuous absorbance measuring device to the tube containing the separated sample, it was confirmed that foot-and-mouth disease virus was contained in the peaks of fractions 7 and 8.
Figure 1c is an immunochromatization method (Western blot) that identifies only specific proteins in a sample. After fixing proteins separated by electrophoresis (SDS-PAGE) on a membrane, chemiluminescence by reacting antibodies that bind to specific proteins As a method of checking the presence or absence of a protein through (chemiluminescence), the sucrose peak fraction containing foot-and-mouth disease virus obtained in FIG. 1b was electrophoresed, and then the residual fraction of the non-structural protein was confirmed using a non-structural protein antibody. As a result, 0.5M sulfuric acid It was confirmed that the foot-and-mouth disease vaccine antigen and the non-structural protein were completely separated at the ammonium concentration.
FIG. 2 shows the detection results of non-structural proteins when goats are inoculated with foot-and-mouth disease vaccine antigens produced under conditions of selectively removing only non-structural proteins using the hydrophobic interaction chromatography method of the present invention.

Hereinafter, the examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, those of ordinary skill in the art to which the present invention pertains. It will be obvious to you.

Example 1. Establishment of conditions for removing non-structural proteins using hydrophobic interaction chromatography

The present inventors confirmed conditions for selectively removing only non-structural proteins using a hydrophobic interaction chromatography method.

First, in order to secure foot-and-mouth disease virus complete particles, BHK21 cells were cultured, and when monolayer formation in the flask was confirmed, foot-and-mouth disease virus (Type O Jincheonju) was inoculated into the cells at a concentration of 0.001 MOI (Multiplicity of Infection). Thereafter, when a cytopathic effect (CPE) was observed at 90% or more, the supernatant was recovered by centrifugation. The virus supernatant was inactivated at 26° C. for 24 hours by adding 3mM binary ethyleneimine as an inactivating agent, and the inactivating agent was neutralized with 2% sodium trisulfate (1N Sodium thiosulfate). 1 ml of the inactivated virus supernatant was used as a sample.

Ammonium sulfate powder was mixed with a 20 mM phosphate buffer solution to prepare an ammonium sulfate solution, and then filtered through a 0.2 μm filter. A hydrophobic interaction column (HiTrap Butyl Fast Flow Column, GE Healthcare) using a butyl group was connected to an AKTA Pure 150 (GE healthcare) Fast Protein Liquid Chromatography purification system. Ammonium sulfate solution was mixed with the inactivated foot-and-mouth disease virus supernatant to a final concentration of 0, 0.25, 0.5, 0.75, and 1.0 M, and then injected into the column, and only 20 mM phosphate buffer solution containing no ammonium sulfate was used for elution. . The flow rate was 1 ml/min, and 2 ml per fraction were harvested.

As a result, as shown in FIG. 1A, the fraction recovered without binding to the column was peak-1, and the fraction eluted after binding to the column was peak-2.

In addition, the two peak fractions were concentrated to quantify the foot-and-mouth disease virus vaccine antigen through sucrose density gradient ultra-fast centrifugation.

As a result, as shown in FIG. 1b, foot-and-mouth disease virus vaccine antigens located in fractions 7 and 8 due to the difference in density gradient were isolated in peak-1 fraction in the groups treated with ammonium sulfate 0, 0.25 and 0.5M, and 0.75 , In the 1.0 M treatment group, it was separated from the peak-2 fraction.

In addition, after protein electrophoresis (SDS-PAGE) of the obtained fractions of sucrose No. 7 and 8 containing foot-and-mouth disease virus, immunoblotting was performed using NSP (3B) antibody to determine non-structural proteins by ammonium sulfate concentration. The eluted fraction was confirmed.

As a result, as shown in Figure 1c, in the ammonium sulfate 0 and 0.25 M treatment groups, the peak-1 fraction was eluted, in the 0.5 and 0.75 M treatment groups, the peak-2 fraction was eluted, and in the 1.0 M treatment group, the ammonium sulfate was eluted. As a result, it was confirmed that non-structural proteins were precipitated and all disappeared before column injection.

Therefore, by varying the degree of hydrophobicity by treatment with each concentration of ammonium sulfate, the concentration conditions for each concentration of the vaccine antigen and the non-structural protein eluted in the peak-1 and peak-2 fractions were set, and the results were synthesized to determine the non-structural protein from the vaccine antigen. Optimal conditions for removal were established.

That is, when treated with 0.5 M ammonium sulfate, foot-and-mouth disease vaccine antigen (foot-and-mouth disease virus complete particle form; 146S) was eluted unbound to the column, and the non-structural protein was mostly bound to the hydrophobic ligand, so that the foot-and-mouth disease vaccine antigen and the non-structural protein were successfully separated.

In addition, as shown in Figure 1c, the degree of chemiluminescence of the peak-1 fraction (8th lane) of the PEG precipitation method (16th lane) and the ammonium sulfate 0.5M treatment group, which are commonly used for the concentration and purification of foot-and-mouth disease vaccine antigens In comparison, the peak-1 fraction of the 0.5 M ammonium sulfate treatment group showed a markedly low luminescence, which means that the non-structural protein was detected at a lower level, so the effect of improving the removal rate of the non-structural protein contained in the vaccine antigen was confirmed. .

In addition, the contents of foot-and-mouth disease virus and non-structural proteins were compared according to the ultrafiltration method, PEG precipitation method, and hydrophobic interaction chromatography method of the present invention, which are commonly used for concentration and purification of foot-and-mouth disease vaccine antigens. The results of comparing the contents of foot-and-mouth disease virus and non-structural protein according to the method of the present invention and the conventional concentration and purification method are shown in Table 1 below.

As a result, the recovery rates of foot-and-mouth disease virus all showed similar levels, but there was a large difference in the non-structural protein content.

That is, when using the method of the present invention, the vaccine antigen is recovered at a level similar to that of the conventional concentration and purification method, and at the same time, the non-structural protein is only present in a trace amount (1.8 ng/ml), but most of it is removed, so the purity of the foot-and-mouth disease vaccine antigen is excellent. Improved.

Way Foot-and-mouth disease vaccine antigen recovery rate Non-structural protein content PEG precipitation method 80% 9.2 ng/dose Ultrafiltration 95% 213 ng/dose Hydrophobic interaction chromatography method of the present invention
(Non-binding elution) 90% 1.8 ng/dose

Example 2. Confirmation of detection of non-structural proteins after inoculation of foot-and-mouth disease vaccine prepared by the method of removing non-structural proteins using hydrophobic interaction chromatography

The present inventors used the hydrophobic interaction chromatography method of the present invention established in Example 1 above to determine the purity of the foot-and-mouth disease vaccine antigen produced under the condition of selectively removing only non-structural proteins, by using chlorine having the highest immunogenicity. After inoculation 5 times (2 ml/dose) as a target, the detection and level of non-structural proteins were confirmed through three different enzyme-linked immunoassay (ELISA) kits.

As a result, as shown in Fig. 2, the minimum content of the non-structural protein (3AB) in which an antibody against the non-structural protein is not detected was measured at a level of 2.6 ng/dose.

This indicates that no antibody was detected in the enzyme-linked immunoassay (ELISA) diagnostic kit even when 2.6 ng/dose of the non-structural protein (3AB) was repeatedly inoculated into goats 5 times (PI≤50).

Therefore, the non-structural protein of 1.8 ng/ml content contained in the foot-and-mouth disease vaccine antigen obtained by the method of the present invention can be assured that no antibody is detected even when inoculated 5 times (2 ml/dose) in goats. Since the result is derived from the goat with the highest origin, it can be ensured that non-structural protein antibodies are not detected even when vaccinating other targets, that is, pigs or cows.

Overall, when the non-bonded elution method is used as the hydrophobic interaction chromatography method of the present invention, it provides higher efficiency in terms of time and economy than the post-bonded elution method performed when using the conventional hydrophobic interaction column. In addition, since only non-structural proteins can be selectively removed in the end, there is no fear of causing confusion in discrimination between infection axis and vaccine axis during serological inspection because non-structural protein antibodies are not induced even if the vaccine is inoculated to the target axis more than 5 times.

Accordingly, the present invention provides an improved method to additionally improve the purity of vaccine antigens while ensuring the vaccine antigen recovery rate of the existing concentration and purification methods, thereby contributing to enhancing the quality competitiveness of the vaccine by utilizing it as a localization technology for foot-and-mouth disease vaccine.

Claims (8)

Domestic type O Jincheonju foot-and-mouth disease virus vaccine antigen preparation method comprising the following steps:
(a) obtaining and inactivating the supernatant of the domestic type O Jincheonju foot-and-mouth disease virus; And
(b) After mixing the inactivated domestic type O Jincheonju foot-and-mouth disease virus supernatant of step (a) and a phosphate buffer solution containing 0.5M ammonium sulfate, a pH of 7.5 to 8.0 and a flow rate of 1.0 ml/min Fast Protein Liquid Chromatography was performed with a hydrophobic interaction chromatography column using a butyl group in Korea, and non-structural proteins (NSPs) were removed. Obtaining a virus vaccine antigen,
A non-structural protein is attached to the ligand of the hydrophobic interaction chromatography column, thereby removing the non-structural protein;
Recovering a fraction not attached to a ligand of the hydrophobic interaction chromatography column, which is a filtrate that has passed through the hydrophobic interaction chromatography column;
It characterized in that obtaining the domestic type O Jincheonju foot-and-mouth disease virus vaccine antigen in complete particle form (146S) from the fraction not attached to the recovered hydrophobic interaction chromatography column ligand,
Domestic type O Jincheonju foot-and-mouth disease virus vaccine antigen production method.
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