JP7186215B2 - Methods of obtaining proteins from BORDETELLA PERTUSSIS including freezing and thawing processes - Google Patents

Description

The present invention relates to a method for purifying PRN protein from Bordetella pertussis, including freezing and thawing processes.

Pertussis is an acute respiratory illness that occurs primarily in infants and is characterized by coughing for two weeks or longer. Bordetella pertussis, a Gram-negative, aerobic short coccobacillus, has been reported to cause whooping cough. Bordetella pertussis uses humans as its sole host and infects primarily through the respiratory tract. In addition, Bordetella pertussis lives on the airway mucosa and causes disease in humans. In the 1930s, a cellular pertussis vaccine was developed and proved to be protective against whooping cough. Additionally, in the 1940s, pertussis vaccines were combined with tetanus and diphtheria inactivated killed vaccines; however, adverse effects of whole-cell pertussis vaccines (epileptic seizures, edema, fever, etc.) were reported. Therefore, the development of a safe pertussis vaccine was needed.

In the 1950s, research into the virulence of Bordetella pertussis was carried out, with components such as pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN) and fimbriae (FIM) as light sources. Reported. Subsequently, development of an acellular pertussis vaccine involving the isolation and purification of these proteins was underway. Since the 1980s, a purified pertussis vaccine has been developed and was vaccinated for the first time in Japan.

To prepare an acellular pertussis vaccine, proteins such as PT, FHA and PRN had to be purified. Conventionally, antigens were co-purified by repeated ammonium sulfate precipitation and density gradient centrifugation. However, such methods have the drawback that many impurities are produced and the purification process is difficult to control. There is another method by which each antigen can be purified individually using a combination of physical and chemical methods. Korean Patent Publication No. 2015-0124973 describes an acellular pertussis vaccine containing PT, FHA, and FIM types 2 and 3.

On the other hand, pertussis vaccine manufacturing processes involving mixing three or four components suffer from the problem that a decrease in the productivity of even one component can lengthen the overall production period.

Therefore, there is a need for a method of efficiently producing large amounts of PRN protein for the efficient preparation of pertussis vaccines.

In this regard, the present inventors discovered that samples containing Bordetella pertussis were subjected to a freezing step (cold shock ) and the thawing step increases the amount of PRN protein extracted from Bordetella pertussis, thereby completing the present invention.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method for obtaining a PRN protein that allows for an increased extraction of Bordetella pertussis PRN protein.

To achieve the above objects, the present invention provides a step of freezing a sample containing Bordetella pertussis; 2) thawing the frozen sample; 3) crushing the thawed sample; A method for obtaining a PRN protein from Bordetella pertussis is provided, comprising the step of purifying the sample.

A method of obtaining Bordetella pertussis PRN protein according to the present invention comprises freezing a sample containing Bordetella pertussis and thawing the sample at refrigeration temperatures such that the amount of PRN protein extracted is maximized. When the PRN protein was obtained by purifying the sample pretreated under the above conditions, the amount of PRN protein extracted was significantly increased compared to the extraction method without such pretreatment. Furthermore, the method of obtaining said PRN protein can also be effectively used for large-scale production of PRN protein for the purpose of producing pertussis vaccines.

FIG. 1 shows a flow chart of the PRN purification process. FIG. 2 shows a detailed flowchart of the process for pellet preparation and thawing and for urea treatment. FIG. 3 shows the results obtained by treating pellets obtained under various preparation and storage conditions with urea. FIG. 4a shows the difference in the amount of PRN protein extracted when PRN protein was obtained by the methods of Comparative Examples 1 to 5 and Examples 1 to 3 using SDS-PAGE technology. FIG. 4b shows the difference in the amount of PRN protein extracted when PRN protein was obtained by the methods of Comparative Examples 6-10 and Examples 4 and 5 using SDS-PAGE technique. FIG. 4c shows the difference in the amount of PRN protein extracted when PRN protein was obtained by each method of Comparative Examples 11 to 16 and Example 6 using SDS-PAGE technology. FIG. 5a shows the difference in the amount of extracted PRN protein obtained by the methods of Comparative Examples 1 to 5 and Examples 1 to 3 using the Western blot technique. FIG. 5b shows the difference in the amount of PRN protein extracted by the methods of Comparative Examples 6 to 10 and Examples 4 and 5 using the Western blot technique. FIG. 5c shows the difference in the amount of PRN protein extracted by the methods of Comparative Examples 11 to 16 and Example 6 using the Western blot technique. FIG. 6 shows the amount of PRN protein extracted when the PRN protein was obtained by the methods of Comparative Examples 1-7 and Examples 1-3 under various slurry and pellet storage conditions. FIG. 7 shows the amount of PRN protein extracted when PRN protein was obtained by each method of Comparative Examples 15 to 17 and Example 7 under various pellet storage conditions. FIG. 8 shows the difference in the amount of PRN protein extracted using different pellet thawing conditions.

In aspects of the invention, 1) freezing a sample containing Bordetella pertussis; 2) thawing the frozen sample; 3) crushing the thawed sample; and 4) purifying the crushed sample. A method of obtaining PRN protein from Bordetella pertussis is provided, comprising steps.

First, a step of freezing the sample containing Bordetella pertussis is performed.

In the present invention, "Bordetella pertussis", also called Bordetella parapertussis, is a Gram-negative coccobacillus of only about 0.3 to 1 μm without flagella and spores. PRN, one of the major proteins of Bordetella pertussis, is an abbreviation for pertactin, the virulence factor of Bordetella pertussis that causes whooping cough. In addition, PRN, an outer membrane protein that adheres to tracheal epithelial cells, is obtained from Bordetella pertussis and is one of the key components of pertussis vaccines.

In the present invention, the "sample containing Bordetella pertussis" refers to a culture obtained by culturing Bordetella pertussis, a slurry separated from the culture by continuous centrifugation, or a slurry obtained from a slurry by high-speed centrifugation. It can be pellets, pellets being preferred. Here, the resulting slurry may be immediately centrifuged to obtain a pellet. However, the resulting slurry may be centrifuged to obtain a pellet after 1-5 days. The slurry may now be stored at 0°C to 25°C. However, the invention is not limited to these.

In an embodiment of the invention, the Bordetella pertussis strain is cultured in modified stainer scholte (MSS) medium at a temperature of 35° C., the cell culture is continuously centrifuged at room temperature for 2 hours, and A pellet was produced by high-speed centrifugation of the resulting slurry at 7,000 rpm for 50 minutes at 2-8°C.

Second, a step of thawing the frozen sample is performed.

In the step of freezing a sample containing Bordetella pertussis strain, freezing is performed at -1°C, -2°C, -5°C, -10°C, -20°C, -30°C, -40°C, -50°C, - at a temperature of 60°C, -70°C, -80°C or -90°C, or -1°C to -90°C, -5°C to -85°C, -15°C to -75°C or -30°C to -60°C at a temperature of freezing for 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 20 hours, 30 hours or 40 hours; 5 hours to 10 hours can be performed.

In addition, thaw slowly by refrigerating frozen samples. wherein thawing is at a temperature of 1°C, 2°C, 3°C, 4°C, 5°C, 7°C, 10°C, 15°C, 20°C or 25°C or C., 5.degree. C. to 15.degree. C. or 7.degree. C. to 10.degree. Thaw for 0.1 hours, 1 hour, 5 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 60 hours, or 0.1 hours to 60 hours, 1 hour to 55 hours, 5 hours to It can be done by refrigerating for 50 hours or 12-48 hours.

In an embodiment of the invention, the pellets were stored frozen at -30°C or below for 2 hours and the frozen pellets were thawed at room temperature or refrigerated temperatures between 1°C and 7°C.

Here, the freezing step and the thawing step can be repeated one or more times.

Third, a step of crushing the thawed sample is performed.

The thawed sample is subjected to the crushing process. Here, crushing can take place, for example, via urea treatment, heat treatment, guanidine treatment and iodine treatment. However, the crushing method is not limited to these, and various crushing methods known in the art can be applied.

If the urea treatment disrupts the sample, the treatment may be at a urea concentration of 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M or 10 M, or from 0.1 M to 10 M, 1 M Urea concentrations of ~8M, 3M-7M or 4M-5M can be performed. Urea treatment is performed for 5 minutes, 10 minutes, 20 minutes, 50 minutes, 80 minutes, 120 minutes, 180 minutes, 240 minutes or 360 minutes, or 5 minutes to 360 minutes, 20 minutes to 240 minutes or 50 minutes to 180 minutes. can break

In an embodiment of the present invention, the thawed pellet was loaded with 4 times the pellet weight of 5.0 M urea buffer and stirred for 3 hours to disrupt the outer membrane of Bordetella pertussis in the pellet.

Fourth, a step of purifying the crushed sample is performed.

After centrifugation of the cell lysate or cell lysate, unwanted cell debris can be removed using various column chromatography methods and the like. Column chromatography methods include ion exchange chromatography, hydrophobic interaction chromatography, gel exclusion chromatography, gel filtration chromatography, HPLC, reverse phase HPLC, affinity chromatography, and the like.

In an embodiment of the present invention, in the step of purifying the crushed pellet, PRN protein is obtained by performing ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). rice field.

As used herein, "ion exchange chromatography" can employ ion exchange resins in which anions or cations are covalently bound to a stationary phase that is the exchange resin. Oppositely charged solute ions in the mobile phase are attracted to the stationary phase by electrostatic attraction. Ion exchange chromatography is based on the equilibrium achieved by the adsorption of ions or charged compounds to ion exchangers by electrostatic forces.

In an embodiment of the present invention, an anion exchange resin is used in an ion exchange chromatography column, a Tris-HCl solution is used as an equilibration buffer, a NaCl-containing Tris-HCl solution is used as a wash buffer, and a NaCl-containing Tris-HCl solution is used as an elution buffer. to elute the PRN protein.

As used herein, the term "hydrophobic interaction chromatography" refers to preparative methods that use hydrophobic interactions between molecules and matrices having hydrophobic functional groups. Hydrophobic functionality can be imparted to the matrix by denaturing the hydrophilic, inert agarose. Modified agarose obtained by reacting alkylamines with BrCN-activated agarose is widely used. Hydrophobic interaction chromatography is widely used for pure separation of proteins. In addition, for hydrophobic interaction chromatography, the ionic strength can be lowered or the pH can be increased for protein elution, using aliphatic amines, alcohols, or nonionic detergents to reduce polarity. (eg, Tween-20 and Triton X-100) can be used.

In an embodiment of the present invention, the PRN protein was eluted using a Tris-HCl solution containing NaCl solution as the equilibration buffer, a Tris-HCl solution containing NaCl solution as the washing buffer, and a Tris-HCl solution containing NaCl solution as the eluent. .

As used herein, "gel filtration chromatography" uses molecular sieves in the stationary phase, which are hydrophilic and therefore can absorb water and swell, Sephadex, polyacrylamide , or agarose gel. If the size of the sample molecule is larger than the largest pore size of the swollen gel, the molecule will not pass through the gel particles and will exit the column through the spaces of the stationary phase particles. Smaller molecules enter the open pores of gel particles and pass at varying speeds depending on their size and shape. Thus, molecules are eluted in order of decreasing size. Gel chromatography takes into account sample size, viscosity, ionic strength, flow rate, and the like.

In an embodiment of the present invention, PRN protein was eluted using sodium sulfate containing NaCl solution as the equilibration buffer.

MODES OF THE INVENTION The invention will now be described in more detail by means of the following examples. However, the following examples are intended only to illustrate the invention and are not intended to limit the scope of the invention.

Example 1. Process to obtain PRN The process to obtain PRN was performed in the following process steps: pellet preparation and thawing, urea treatment, column chromatography (IEX, HIC and SEC), concentration and buffer exchange (UF/DF), and impurities. Purification of free PRN protein. A flowchart of the process of obtaining a PRN is shown in FIG.

Example 1.1. Cultivation of Bordetella pertussis Bordetella pertussis (Korea National Institute of Health) was cultured in MSS medium at a temperature of 35°C for 4 days. Specifically, the culture period was 1 day for the seed culture, 1 day for the primary concentration culture, 1 day for the secondary concentration culture, and 1 day for the main culture.

Example 1.2. Pellet preparation Cell cultures were separated into cell supernatant and slurry by continuous centrifugation at room temperature for 2 hours at a flow rate of 100 L/h and 9,500 rpm. The collected slurry was centrifuged at high speed at 7,000 rpm for 50 minutes at 5° C.±3° C. to obtain a pellet. In the transfer examples, where the slurry was stored for 3 days, the slurry was stored at a refrigerated temperature of 5°C for 3 days and then high speed centrifuged to prepare a pellet.

Example 1.3. Freezing and thawing Prepared pellets were stored at -30°C or below for 2 hours, then stored at room temperature (RT) for 1 day and thawed. The PRN protein was then obtained by following the procedures of Examples 1.4-1.7 below. Furthermore, experiments were conducted by changing the slurry storage conditions and freezing and thawing conditions as in Examples 1 to 6 and Comparative Examples 1 to 16 shown in Table 1 below, and then Examples 1.4 to 1.1 below. 7 procedures were performed to obtain the PRN protein.

Example 1.4. Urea Treatment To the thawed pellet was added 4 times the weight of the pellet of 5.0 M urea buffer; stirring was performed using a magnetic bar at a speed of 280 rpm for 3 hours. The urea-treated solution, which had been stirred for 3 hours, was subjected to high speed centrifugation at 7,000 rpm (12230 RCF) at 5°C ± 3°C for 1.5 hours. After this centrifugation, cell debris was removed and the supernatant was collected. The recovered supernatant was filtered through a 0.22 μm filter. A detailed process flow chart of pellet preparation, thawing of Examples 1.2 and 1.3, and urea treatment is illustrated in FIG.

Example 1.5. Purification by IEX Column A column packed with IEX resin was flushed with 3 CV of distilled water (DW) at 100 cm/hr to remove the stock solution. Then, the column was washed by flowing a 1N NaOH solution at 40 cm/hour for 1 hour. The column was equilibrated by running 3 CV of an equilibration buffer, Tris-HCl. After equilibration was complete, the process solution for concentration and buffer exchange 1 was run through the IEX column for adsorption. The column was flushed with 3 CV of the equilibration buffer to remove any remaining solution in the column. Thereafter, the column was washed by running 5 CV of a Tris-HCl solution containing a NaCl solution as a washing buffer. After washing was completed, 5 CV of a Tris-HCl solution containing an NaCl solution, which is an elution buffer, was run through the column to elute the PRN protein.

Example 1.6. Purification by HIC column The stock solution was removed by running 3 CV of distilled water at 100 cm/h through the column packed with HIC resin. Then, the column was washed by flowing a 1N NaOH solution at 40 cm/hour for 1 hour. The column was equilibrated by running 3 CV of a Tris-HCl solution containing an NaCl solution as an equilibration buffer. After completion of equilibration, the solution obtained by mixing the eluent obtained through the purification process with the IEX column and 3.6M NaCl at a ratio of 1:1 was passed through the HIC column for adsorption. The column was flushed with 3 CV of the equilibration buffer to remove any remaining solution in the column. Thereafter, the column was washed by running 5 CV of a Tris-HCl solution containing a NaCl solution as a washing buffer. After washing was completed, 5 CV of a Tris-HCl solution containing an NaCl solution as an elution buffer was run to elute the PRN protein.

Example 1.7. Purification by GFC column The stock solution was removed by running 3 CV of distilled water at 15 cm/h through the column packed with GFC resin. Then, the column was washed by flowing a 1N NaOH solution at 15 cm/hour for 1 hour. Equilibration was carried out by running 3 CV of sodium phosphate containing NaCl solution as equilibration buffer. After equilibration was complete, the process solution for concentration and buffer exchange 2 was run over the GFC column to elute the PRN protein.

Experimental example 1. Identification of Differences in PRN Extraction Amount Depending on Pellet Preparation and Storage Conditions Experiments were conducted by changing the pellet preparation and storage conditions in the PRN acquisition process of Example 1 in order to identify conditions that effectively increased the amount of PRN protein extracted. rice field. The pellet preparation and storage conditions are as shown in Table 1 of Example 1.3, and the experimental procedures and results under each condition are shown in Experimental Examples 1.1 to 1.5 below.

Experimental example 1.1. Color change of supernatant caused by urea treatment due to slurry and pellet storage conditions Each pellet obtained by using different preparation and storage conditions as shown in Table 1 of Example 1.3 was treated with urea buffer. , stirring was performed. The result was then centrifuged. The supernatant obtained by centrifugation was filtered, and the difference in color of the supernatant due to each storage condition was identified. The above process is detailed in Example 1.4 and experimental results are shown in FIG.

As shown in Figure 3, the supernatant was dark yellow in color when the pellet was cryopreserved and then refrigerated (Examples 2 and 3, and Examples 5 and 6). showed that. This means that many pellets are crushed when treated with urea after cryopreservation and refrigeration.

Experimental example 1.2. Identification of difference in protein extraction amount due to pellet storage conditions SDS-PAGE and Western blotting techniques were used to identify differences in PRN protein extraction amount due to pellet storage conditions. Here, guinea pig anti-PRN (Young In Frontier) and biotin-labeled guinea pig anti-PRN (Young In Frontier) were used as antibodies in the Western blot technique. These results are shown in FIGS.

As shown in Figures 4 and 5, cryopreservation of the pellet followed by refrigeration yielded large amounts of PRN protein.

Experimental example 1.3. Identification of Extractable Amounts of PRN Protein by Slurry and Pellet Storage Conditions To identify differences in the amount of PRN protein extracted by slurry and pellet storage conditions, an enzyme-linked immunosorbent assay (ELISA) technique was used. These results are shown in FIG. Here, the pellet storage conditions for each of the numbers 1 to 11 at the bottom of the graph in FIG. 6 are as follows, and more detailed conditions are shown in Table 2 below:
1: Urea treatment immediately after pellet preparation;
2: Cryopreserved and thawed at room temperature for 1 day;
3: Thaw at room temperature for 1 day without cryopreservation;
4: Store frozen and thaw in refrigerator for 1 day;
5: Cryopreserved and thawed in refrigeration for 3 days;
6: Thaw in refrigeration for 1 day without freezing;
7: Thaw in refrigeration for 3 days without freezing;
8: Cryopreserved for 1 day (-30°C);
9: 3 days frozen storage (-30 ° C);
10: 1 day cryopreservation (-70°C); and 11: 3 days cryopreservation (-70°C).

As shown in Figure 6, pellets prepared after 0 days of storage in slurry state showed an increase in the amount of PRN protein extracted over time during the time the pellets were cryopreserved (Examples 2 and 3). pellets prepared after 3 days storage in slurry state showed a decrease in extractable amount of PRN protein over time during pellet cryopreservation (Examples 5 and 6); by comparison with). Furthermore, cryopreserved pellets showed increased extraction of PRN protein compared to non-cryopreserved pellets (Example 1 vs. Comparative Example 2, Example 2 vs. Comparative Example 3). and comparison between Example 3 and Comparative Example 4). This is because, in order to increase the amount of PRN protein extracted, it is more effective to immediately pellet the slurry and store the pellet than to store the slurry as it is. This means that the amount of extraction of

Experimental example 1.4. Identification of differences in the amount of PRN protein extracted by pellet thawing In FIG. In Comparative Example 13, Comparative Examples 6 and 14, Comparative Examples 7 and 15, and Comparative Examples 8 and 16), the experimental groups that were frozen and then thawed (Example 1 and Example 4, Examples 2 and 5, and Examples 3 and 6) showed a significant increase in the amount of PRN protein extracted. Therefore, further experiments were performed to identify whether the amount of PRN protein extracted varies upon thawing. Here, the thawing of the pellets was carried out at a refrigeration temperature of 5°C for 48 hours. Experimental results are shown in Table 3 and FIG.

As shown in Table 3 and Figure 7, approximately a 3-fold increase in the amount of PRN protein extracted was observed when the pellet was thawed prior to urea treatment compared to when the pellet was frozen. Low levels of PRN protein were extracted in all three experimental groups in which the frozen pellets were urea treated. This means that thawing the pellet before urea treatment has a great effect on PRN protein extraction. These results indicated that the pellet had to be thawed prior to urea treatment in order to effectively increase the amount of PRN protein extracted.

Experimental example 1.5. Identification of differences in the amount of PRN protein extracted by pellet thawing conditions In order to identify differences in the amount of PRN protein extracted by the pellet thawing method, the pellets were cryopreserved and then refrigerated (2 days) and room temperature (1 hour), respectively. ), and the amount of PRN protein extracted when stored at 37° C. (15 minutes). These results are shown in FIG.

As shown in FIG. 8, when thawing was carried out, when thawing gradually with refrigeration, an increase in the amount of PRN protein extracted was observed compared to when thawing rapidly at high temperature. In addition, approximately a two-fold increase in the amount of PRN protein extracted was observed when thawed in the refrigerator for 2 days compared to thawed at 37°C for 15 minutes. These results demonstrate that the pellet must be slowly thawed in a refrigerator after freezing in order to effectively increase the amount of PRN protein extracted.

Experimental example 2. Identification of the possibility of scale-up In order to identify whether the conditions that effectively increase the extraction amount of PRN protein identified in Experimental Examples 1.1 to 1.5 can be applied even at the 50 L scale, slurry was immediately pelleted without storage, the resulting pellet was stored frozen (-30°C) for 1 day and then thawed in a refrigerator (5°C) for 2 days. Afterwards, 533 g of pellet was recovered, treated with 1,800 mL of 5.0 M urea buffer and stirred for 3 hours using a magnetic bar. The stirred urea-treated solution was centrifuged at a speed of 6,300 rpm for 1.5 hours. After centrifugation, cell debris was removed; 1,650 mL of supernatant was collected and filtered. The procedures of Examples 1.5 to 1.7 were then followed. Results are shown in Table 4.

As shown in Table 4, the conditions identified in Experimental Examples 1.1 to 1.5 that effectively increase the amount of extracted PRN protein can be applied even at the 50 L scale, and approximately 300 mg of PRN protein can be extracted at the 200 L scale. It was found that ~400 mg could be obtained.
This application also includes the following aspects.
[Aspect 1]
A method for obtaining PRN protein from Bordetella pertussis, comprising:
1) freezing a sample containing Bordetella pertussis;
2) thawing the frozen sample;
3) crushing the thawed sample; and
4) Step of purifying the crushed sample
A method, including
[Aspect 2]
A method according to aspect 1, wherein the sample containing Bordetella pertussis is a pellet obtained by centrifuging a Bordetella pertussis culture.
[Aspect 3]
A method according to aspect 1, wherein in step 1) the freezing is carried out at a temperature of -5°C to -85°C.
[Aspect 4]
A method according to aspect 1, wherein in step 1) the freezing is carried out for 20-30 hours.
[Aspect 5]
A method according to aspect 1, wherein in step 2) the thawing is carried out in refrigeration.
[Aspect 6]
A method according to aspect 5, wherein the thawing is carried out by refrigerating at a temperature of 1°C to 10°C.
[Aspect 7]
A method according to aspect 5, wherein thawing is carried out by refrigerating for 36-48 hours.
[Aspect 8]
The method according to aspect 1, wherein in step 3), the crushing is performed by any one treatment selected from the group consisting of urea treatment, heat treatment, guanidine treatment and iodine treatment.
[Aspect 9]
A method according to aspect 8, wherein the urea treatment is performed at a urea concentration of 3M to 7M.
[Aspect 10]
A method according to aspect 8, wherein the urea treatment is carried out for 150-210 minutes.
[Aspect 11]
A method according to aspect 1, wherein in step 4) the purification is performed by performing ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC).

Claims (9)

A method for obtaining PRN protein from Bordetella pertussis, comprising:
1) freezing a sample containing Bordetella pertussis;
2) thawing the frozen sample;
3) crushing the thawed sample; and 4) purifying the crushed sample ,
A sample containing Bordetella pertussis is obtained by centrifuging the Bordetella pertussis culture to obtain a slurry, and then centrifuging the slurry immediately or by storing the slurry at 5° C. for 1-3 days before centrifuging. is the resulting pellet,
In step 1), freezing is performed at a temperature of −30° C. or lower for 20 hours to 10 days,
In step 2), thawing is carried out at a temperature of 1° C. to 20° C. with refrigeration for 12 hours to 60 hours.
How . The method according to claim 1, wherein in step 1) the freezing is carried out at a temperature of -30°C to -85°C. The method according to claim 1 or 2 , wherein in step 1) freezing is carried out for 20-30 hours. A method according to any one of claims 1 to 3 , wherein thawing is carried out by refrigerating at a temperature of 1°C to 10°C. A method according to any one of claims 1 to 4 , wherein thawing is carried out by refrigerating for 36-48 hours. The method according to any one of claims 1 to 5, wherein in step 3), crushing is performed by any one treatment selected from the group consisting of urea treatment, heat treatment, guanidine treatment and iodine treatment. 7. The method of claim 6 , wherein the urea treatment is performed at a urea concentration of 3M-7M. A method according to claim 6 or 7 , wherein the urea treatment is carried out for 150-210 minutes. Purification in step 4) is carried out by performing ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC), according to any one of claims 1 to 8. the method of.

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