KR100405174B1 - Purification method of surface antigen of recombinant B-type hepatitis virus - Google Patents

Abstract

The present invention comprises the steps of (a) crushing the cells using a high pressure of 15,000 to 20,000 psi to remove cell debris; (b) concentrating and pH precipitation after diafiltration into a large amount of buffer using a Millipore Pelican 2 cassette 300K membrane; (c) ultracentrifuging for 24 to 48 hours using a Beckman L7-35 ultracentrifuge manufactured by Beckman at Rotor 45Ti, rotational speed of 40,000 to 45,000 rpm; And (d) passing the gel filtration chromatography using a Sephacryl S-300 or Sephacryl S-400 column of Pharmacia Biotech Co., Ltd., to a method for purifying recombinant hepatitis B virus surface antigen. According to the present invention, it is possible to provide a method for purifying recombinant hepatitis B virus surface antigen which can improve production yield and reduce manufacturing cost through improvement and simplification of the process.

Description

Purification method of surface antigen of recombinant B-type hepatitis virus

The present invention relates to a method for purifying recombinant hepatitis B virus surface antigen (HBs antigen). More specifically, the present invention relates to a method of purifying HBs antigen expressed in recombinant yeast with high purity after cell disruption, concentration and diafiltration through pH precipitation, ultracentrifugation and gel filtration chromatography.

Hepatitis B is a viral disease commonly found mainly in tropical Africa, Southeast Asia, and the Far East. It is infectious and can cause acute and chronic hepatitis, cirrhosis and liver cancer. More than 1 billion people are infected with the hepatitis B virus worldwide, and it is estimated that more than 2 million people die each year from hepatitis B and its sequelae. Korea is a pandemic region of the hepatitis B virus, and 3 to 10% of the population is carriers, and more than 70% of Korean chronic hepatitis, cirrhosis and liver cancer are caused by chronic infection of the hepatitis B virus.

In general, three types of antigens are found in the plasma of hepatitis B carriers: spherical particles having a diameter of 22 nm, cylindrical particles having the same diameter, and virus particles having a diameter of 42 nm called Dane particles. Virus particles 42 nm in diameter are composed of an envelope and a nucleocapsid 27 nm in diameter containing HBV DNA and DNA polymerase therein. Spherical and cylindrical particles 22 nm in diameter contain no nucleus and consist only of HBs antigen. There are four types of subtypes of adr, adw, ayr, and ayw in HBs antigen particles. Subtypes are known to be specific to ethnic groups.

In 1967, Krugman et al. Confirmed that heat treatment of HBV-containing patients, MS-2, known as MS-2, resulted in loss of infectivity. Moreover, when these patients were vaccinated with infectious original serum, hepatitis It was confirmed that the onset was prevented or reduced. In 1975, Purcell et al. Confirmed the safety and efficacy of inactivated purified hepatitis antigens using chimpanzees. These studies have led to the development of hepatitis B vaccines based on purified antigens isolated from plasma of hepatitis B carriers, and some companies have been using B hepatitis B virus surface antigens purified from plasma. Hepatitis vaccines have been developed. However, despite the merits of human plasma-derived hepatitis B vaccine, it has recently been limited due to the limited supply and demand of raw materials and the possibility of contamination of deadly infectious substances such as AIDS virus. It is showing.

In order to overcome the disadvantages of the plasma-derived hepatitis B vaccine, a yeast-derived hepatitis B vaccine using genetic recombination has been developed. In 1987, several genetically engineered hepatitis B vaccines appeared in the domestic and global markets, led by SmithKlein Beecham, Ltd.'s Engerix B. The world's two largest vaccine makers, Belgium's Smithcline Non-Tragedy, Merck Co. Ltd. in the US, and LG Chem in Korea, have developed a hepatitis B vaccine using Saccharomyces cerevisiae as a vaccine production strain. Develop and sell. In late 1980, a Hansenula polymorpha yeast strain was introduced to produce hepatitis B vaccine, which can significantly increase intracellular protein expression compared to Saccharomyces cerevisiae. I am using the system. These recombinant hepatitis B vaccines, which contain the S-region of the surface antigens of hepatitis B virus, have already been established to be safe and effective and are increasingly being replaced by plasma-derived hepatitis B vaccines worldwide. have.

In order to vaccinate recombinant HBs antigen proteins expressed by yeast cells, related companies have developed a unique purification method for removing impure proteins derived from recombinant organisms.

Patent No. 38837 discloses a step of crushing a culture using glass beads; Separating surface antigens with an affinity column; And purified by CsCl ₂ concentration gradient ultracentrifugation; hepatitis B virus surface antigen was purified through.

Patent Registration No. 65305 discloses the steps of crushing yeast cells; Removing cell debris; Acid treatment of the supernatant to lower the pH, removal of the resulting precipitate, alkali treatment of the supernatant to raise the pH, and then the supernatant collected for microfiltration; Concentrating by ultrafiltration; Recovering the surface antigen and the surface antigen before hepatitis B with carbonate buffer after adsorption by silica treatment; Passing through the anion exchange resin; Hepatitis B surface antigen was purified through a step of passing through hydrophobic chromatography and / or gel filtration chromatography.

Patent No. 103201 discloses lysing Pichia pastoris yeast cells in the presence of chaotropic salts; Separating the hepatitis B virus surface antigen-containing supernatant from the lysed cells; Precipitating fat and contaminating proteins in the supernatant; Diafiltration; Contact with silica, followed by washing with a buffer of pH 6-8, eluting with a buffer eluate of pH 9.5-11.0 containing urea at 0.5-8 molarity; Gel filtration step; And passing through anion exchange chromatography; hepatitis B virus surface antigen was purified through.

Patent Registration No. 159716 discloses a yeast cell disruption step; And supernatant was added to silica to adsorb the HBs antigen and to desorb the HBs antigen.

Patent Registration No. 177254 discloses a surface membrane of a cell membrane by crushing it with a buffer solution containing chaotropic salt in the process of crushing and purifying recombinant cells expressing the whole S2 peptide-containing hepatitis B virus surface antigen. Extracting from; Increasing the pH of the cell extract to increase solubility of the surface antigen and promote particle formation; Precipitating and removing cell debris and contaminants; Adsorption to silica resin; Passing through hydrophobic chromatography; And passing through gel filtration chromatography; hepatitis B virus surface antigen was purified through.

Patent No. 194247 discloses a step of lysing yeast cells in the presence of a surfactant and then separating the supernatant; Contacting the supernatant with silica, and then desorbing the HBs antigen with a buffer of pH 8.8 to 11.0 containing sodium deoxycholate; Adjusting the HBs antigen-containing solution to pH 7-9 and passing it through an anion exchange resin with addition of 0.5 M or less salt; Passing through diafiltration and gelfiltration chromatography; Treating the HBs antigen-containing fraction with proteolytic enzymes; Diafiltration and concentration step; And passing through gel filtration chromatography.

Patent No. 251015 discloses a step of lysing yeast cells in the presence of a surfactant and then separating the supernatant; Contacting the supernatant with silica and desorbing the HBs antigen with a buffer of pH 8.8 to 11.0; Passing through anion exchange resin and eluting with 0.05-0.5 M sodium chloride buffer; And passing through gel permeation chromatography.

However, in the production of recombinant hepatitis B virus surface antigen using such yeast, the conventional methods have raised problems such as the possibility of protein denaturation or endotoxin contamination during the purification process through several steps of purification process.

Concerning Endotoxin Contamination Problems During the Purification of Hepatitis B Virus Surface Antigen, Patent Registration No. 180237 discloses a solution of hepatitis B virus surface antigen solution purified from recombinant yeast with chelating agent and a surfactant to remove endotoxin. Next, this was filtered using a polymer membrane, and the residual solution was diafiltered with a phosphate solution of alkaline pH to remove a chelating agent and a surfactant. However, for this purpose, too many processes are required for purification, thereby lowering productivity.

An object of the present invention is to provide a method for purifying recombinant hepatitis B virus surface antigen which can improve production yield and reduce manufacturing cost through improvement and simplification of the process.

Figure 1 shows the results of analyzing each band obtained in ultracentrifugation by a reverse passive hemagglutination assay (RPHA).

FIG. 2 shows the results of staining with Bandage Brilliant Blue reagent after each band obtained in ultracentrifugation by SDS-PAGE.

Purification method of the hepatitis B virus surface antigen expressed from recombinant yeast cells according to the present invention comprises the steps of (a) crushing the cells using a high pressure of 15,000 to 20,000 psi to remove cell debris; (b) concentrating and pH precipitation after diafiltration into a large amount of buffer using a Pelicon 2 cassette 300K membrane from Millipore; (c) ultracentrifuging for 24 to 48 hours using a Beckman L7-35 (Beckman L7-35) ultracentrifuge at Rotor 45Ti, rotational speed 40,000 to 45,000 rpm; And (d) passing the gel filtration chromatography using a Sephacryl S-300 or Sephacryl S-400 column from Pharmacia Biotech; It includes.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Purification method of hepatitis B virus surface antigen expressed from recombinant yeast cells according to the present invention consists of cell crushing, concentration and diafiltration, followed by pH precipitation, ultracentrifugation and gel filtration chromatography.

The purification method of the present invention is effective for purifying HBs antigen from a culture of any transformed yeast capable of expressing HBs antigen. Transformants include Candida, Kloeckera, Saccharomyces, Schizosaccharo myces, Rhodotorula, Hansenula, Torulopis, Peachia and Clube Vero It may be selected from the group consisting of yeast belonging to the genus Kluyveromyces, these are exemplary and the present invention is not limited thereto.

After floating the yeast cells in a buffer containing urea or chemicals, surfactants and chelating agents having effective urea or equivalent effects, in a short time for effective cell disruption and removal of substances free from the crushed cells, Microfluidics International Inc. Pass through the Microfluidizer from Microfluidics International Co. High pressure is applied to disrupt cells while tracking cell disruption. The preferred concentration of urea is 3 to 6 moles, tween 20 of 0.2 to 0.6% (v / v) is used as a surfactant, and the use of EDTA, EGTA, etc. at a concentration of 15 to 50 mM is used as a chelating agent. desirable. The same procedure is repeated several times until the cells are sufficiently disrupted to effectively break down the cell walls of the yeast and allow the particles composed of hepatitis B virus surface antigens to be released from the cells.

The cell lysate is centrifuged to remove the unbroken cells and debris, the supernatant is taken, concentrated in a Pellicon system at low temperature, and then filtered with phosphate buffer. Millipore's Pelican 2 cassette 300K membrane can be used to effectively remove small-molecular-weight proteins derived from culture media or cells, including chemicals such as surfactants and chelating agents used in cell disruption.

The concentrated and diafiltered solution is adjusted to pH 10 to 10.5 with sodium hydroxide solution, and then the temperature is raised to 30 to 40 ° C. and left for 10 to 20 minutes. It is then quenched with ice water and left to stand until the temperature is between 5 and 10 ° C. The pH is 5.0-5.5 with hydrochloric acid solution on the stirrer, then the precipitate is removed by centrifugation and the pH is adjusted to 9.0-9.5.

The solution was filtered through a sterile filter, and potassium bromide (KBr) was added step by step to prepare solutions having different specific gravity, and then concentration gradient ultracentrifugation was performed. After centrifugation for 20 to 48 hours, only the band containing hepatitis B virus surface antigen is recovered and filtered with a sterile filter. After filtration, the solution is passed through gel filtration chromatography, using either Sephacryl S-300 or Sephacryl S-400 columns.

The method for purifying hepatitis B virus surface antigen expressed from recombinant yeast cells according to the present invention simplifies the steps by not introducing these processes as compared to the conventional techniques using silica gel, anion exchange resin or hydrophobic chromatography during the purification process. In particular, the removal of endotoxin, which is a technical task of some patents, could be achieved without a separate process.

Hereinafter, preferred embodiments and comparative examples of the present invention will be described.

The following examples are intended to illustrate the invention and should not be understood as limiting the scope of the invention.

Example 1

Step 1. Cell Crushing

After incubation, the yeast cells recovered by centrifugation were washed once with distilled water, and then suspended in a cell disruption solution. The cell suspension was placed in a microfluidizer and passed through a high pressure of 15,000 to 20,000 psi. The whole process was performed in the chilled state to maintain the low temperature state, and after each passage, it was left for 10 to 30 minutes to lower the temperature of the raised solution. After crushing three times, the crushed cells were recovered by centrifugation and again suspended in a cell crushing solution to carry out crushing. Cell disruption was determined by microscopic observation and increase in free protein. In addition, the results of Table 1 were obtained by analyzing the change of the amount of free protein according to the number. As shown in Table 1, the rate of increase of free protein decreased with each passage.

Shred Recovery Protein amount (mg) Increase in mg 1-0 3801 1-1 19267 15466 1-2 26956 7689 1-3 31753 4796 2-0 1546 2-1 4221 2674 2-2 6291 2070 2-3 8431 2140

Step 2. Concentration, Diafiltration and pH Precipitation

The cell lysate was centrifuged to obtain a supernatant, and then concentrated by diafiltration with sodium phosphate buffer. The centrifuge supernatant was placed in a Pelicon system equipped with a MWCO 300K membrane, followed by continuous diafiltration using 20-30 times the volume of sodium phosphate buffer.

The concentrated and diafiltered solution was adjusted to pH 10.2 with sodium hydroxide solution, and then the temperature was raised to 37 ° C. and left for 10 to 20 minutes. It was then quenched with ice water and left to stand until the temperature reached 5-8 ° C. The pH was 5.2 using a hydrochloric acid solution on a stirrer, then the precipitate was removed by centrifugation, the pH was readjusted to 9.2 and filtered through a sterile filter.

The diafiltration process was able to remove about 50% of the cell-derived protein and cellular debris, and reduced the total volume by half. The pH precipitation process allowed about 44% of the total protein. Could get

Step 3. Ultracentrifugation

Potassium bromide was added to the supernatant after pH precipitation to make several solutions within the range of specific gravity 1.06 to 1.30 and placed in order in the ultracentrifuge tube. Using Beckman's Beckman L7-35 ultracentrifuge, separation was performed for 24 to 48 hours at Rotor 45Ti and rotational speed of 40,000 to 45,000 rpm to obtain several bands separated according to specific gravity. For each band, the amount of antigen was analyzed using the RPHA kit, and the results are shown in FIG. 1. If small circular spots form at the center bottom of the microplate, it is determined that there is no antigen. In other words, the greater the dilution rate at which no spot is formed, the higher the concentration of the antigen.

Step 4. Gel Permeation Chromatography

The band obtained in step 3 was dialyzed with phosphate buffer, followed by SDS-PAGE. The result is shown in FIG. Impurities other than particles composed of hepatitis B virus surface antigen could be effectively removed by gel filtration chromatography, and the purity of hepatitis B surface antigen in the final stock solution was more than 95%.

Experimental Example 1. Concentration of Endotoxin after Purification

The concentration of bacterial endotoxin was analyzed using the LAL kit (QCL-1000, Bio Whitaker) for the ultracentrifugation and gel filtration chromatography. Table 2 shows the concentrations of bacterial endotoxin (EU / mg) obtained by performing four experiments.

Bacterial Endotoxin Concentration (EU / mg) One 3.06 2 5.6 3 2.18 4 0.88

As shown in Table 2 above, the bacterial endotoxin concentration (EU / mg) was 6 EU / mg or less, which could be used as an injection without further purification. In addition, it was found that the endotoxin was effectively removed, compared to the bacterial endotoxin concentration (per mg of protein) of 8 EU or less in the final crude liquid treated by the conventional purification method (Patent No. 180237).

Therefore, according to the present invention, there is an effect of providing a method for purifying recombinant hepatitis B virus surface antigen, which can improve production yield and reduce manufacturing cost through improving and simplifying the process.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and such modifications and modifications are within the scope of the appended claims.

Claims (3)

(a) disrupting cells and removing debris using a high pressure of 15,000 to 20,000 psi; (b) concentrating and pH precipitation after diafiltration into a large amount of buffer using a Millipore Pelican 2 cassette 300K membrane; (c) ultracentrifuging for 24 to 48 hours using a Beckman L7-35 ultracentrifuge manufactured by Beckman at Rotor 45Ti, rotational speed of 40,000 to 45,000 rpm; And (d) passing the gel filtration chromatography using a Sephacryl S-300 or Sephacryl S-400 column of Pharmacia Biotech Co., Ltd., characterized in that the hepatitis B virus surface expressed from recombinant yeast cells. Method for Purifying Antigens. The method of claim 1, The pH precipitation step adjusts the pH to pH 10 to 10.5, raises the temperature to 30 to 40 ° C. and leaves it for 10 to 20 minutes, then quenchs with ice water, sets the pH to 5.0 to 5.5, and then precipitates the precipitate by centrifugation. Purifying the hepatitis B virus surface antigen expressed from recombinant yeast cells, characterized in that the step of removing, and adjusting the pH to 9.0 to 9.5. The method of claim 1, The ultracentrifugation step was performed using potassium bromide so as to have a specific gravity of 1.06 to 1.30, and centrifugation for 20 to 48 hours to separate the band containing the hepatitis B virus surface antigen. Method for Purifying Hepatitis B Virus Surface Antigen.