KR101632635B1 - Lytic proteins of vero cell, preparation method thereof and detection kit for vero hcp comprising the same - Google Patents

Abstract

The present invention relates to a method for culturing and harvesting a soluble protein of Vero cells, and Vero cells; Destroying and dissolving Vero cells; Purifying the soluble protein of Vero cells; And concentrating to obtain a soluble protein of Vero cells. The soluble proteins of Vero cells prepared according to the method of the present invention have a wide range of applications. They have high sensitivity and good repeatability and can be used for quality control and analysis in the production of vaccines using Vero cells. The present invention also provides Vero cell HCPs assay kits with strong specificity, high sensitivity, and excellent repeatability. This can be used for quality control and analysis in the production of non-secreted virus vaccines against viruses such as hepatitis A virus, as well as other vaccines produced using Vero cells.

Description

TECHNICAL FIELD [0001] The present invention relates to a soluble protein of Vero cells, a method for producing the same, and an assay kit for determining Vero cell HCP comprising the same,

The present invention relates to biotechnology fields, in particular, soluble proteins of Vero cells, a method for producing the same, and an assay kit for determination of Vero cell HCPs using the same.

Bero cells (African green monkey kidney cells) are ideal substrates for vaccine production. Vero cells have a well-defined genetic background and stable karyotype, are free of contamination by exogenous agents and are suitable for large-scale cultivation and production using bioreactors, ensuring homogeneity and stability in mass production of vaccines. Over the years, various vaccines produced with Vero cells, including purified rabies vaccines for human use, inactivated (purified) polio vaccines (IPV), and Japanese encephalitis B vaccines (inactivated) And was licensed for sale in the world market. The production of vaccine using Vero cells can be performed in two ways. The first method is due to the cultivation of secretory viruses, and after the replication is terminated, the virus is secreted into the extracellular culture medium. During incubation of secretory viruses, the virus causes damage to the host cell's structure to exploit the intracellular environment for virus multiplication, which results in the secretion of large amounts of host cell structural proteins into the culture medium following cell death and cell destruction . On the other hand, in response to the demands of cell growth and reproduction, Vero cells will secrete large amounts of cell growth factors into their culture medium during their growth and reproduction processes. The second approach is due to the cultivation of non-secreted viruses, which will be cultured in host cells and remain inside the host cells after viral replication is terminated. During the incubation of non-secreted viruses such as hepatitis A virus in host cells, viral replication will not cause pathological damage to host cells. Thus, the cells need to be destroyed using physical or chemical methods to secrete the virus. In the harvested virus solution, in addition to the cell growth factors secreted during cell growth and proliferation, there are a large amount of cytolytic proteins obtained from cell destruction, which are more complicated components.

Over the years clinical applications have demonstrated the stability and efficacy of Vero cells as a substrate for vaccine production. However, on the one hand, the risk of theoretically possible tumorigenicity of vaccines produced from Vero cells can not be ruled out. There are two measures to control this risk: one is by controlling the passage of Vero cells from 130 to 150 generations, and the other is by reducing the residual DNA and residual protein content of Vero cells. Quality control is still indirectly performed with respect to the total content of vaccine protein, since no specific detection means for Vero cell protein has yet to exist worldwide. (Gao Enming et al., Considerationon the Residual Substances in Vero Cell Vaccines, Center for Drug Evaluation, the State Food and Drug Administration of China, January 12 th, 2007). Under these circumstances, certain components in the remaining host cell proteins (HCPs) can be allergic antigens in vaccines. In 1998, the World Health Organization (WHO) enacted the "Protocol for Production of Biologics Using Animal Cells as Cell Substrate," which calls for an acceptable reduction in the HCP content in consecutive passaged cell lines. This indicates that WHO is beginning to worry about its impact on HCP content and vaccine stability.

Tianbo and Dingziphen reported a method for the quantitative determination of Vero cell HCPs in vaccines (Tian Bo, Ding Zhifen, Chinese Journal of Biologics , 2005, 18 (2): 159-161,164). Wang Hui et al also reported an assay for the residual protein of Vero cells in vaccine manufacture (Wang Hui, Zhang Yuelan, Guo Qinyuan et al., Chinese Journal of Biologics , 2007, 20 (2): 937-939, 947). However, the method of determination for Vero cell residual protein described in the above publications concentrates only on detection of the remaining host protein in the vaccine prepared from the cultivation of the secretory virus, so that the remaining host in the vaccine prepared from the culture of non- It is not suitable for the detection of proteins. Thus, the method for detecting Vero cell HCPs in vaccines as disclosed in the above document still has problems.

Today, no assay kits for detecting the residual content of Vero cell HCPs globally are yet commercially available, the main reason being that it is difficult to produce some of the substances required to determine HCP residual content to be. One of these materials is the soluble protein of Vero cells, which can be used as an immunogen to immunize an animal producing an antibody that can be used for subsequent assay reactions and can be provided as an assay standard in the determination process.

It is an object of the present invention to provide a soluble protein of Vero cells and a process for producing the same. This protein can be used as a standard and an antigen in the analysis of Vero cell HCPs to determine the residual content of Vero cell HCPs in vaccine production and quality control processes.

Another object of the present invention is to provide an assay kit for crystals containing soluble proteins of Vero cells, aiming at overcoming the problems associated with the conventional determination method of Vero cell HCPs.

In order to achieve the above object, in one aspect, the present invention provides a method for producing a bell-cell lysis protein, comprising the steps of:

a) culturing and harvesting Vero cells;

b) destroying and dissolving Vero cells;

c) purifying the soluble protein of Vero cells; And

d) concentrating to obtain a soluble protein of Vero cells.

Bero cell cultivation and harvesting :

The used Vero cells are continuous cell lines of approved and approved monkey kidney cells for the production of vaccines. The vaccines for human use include purified rabies vaccines for human use, inactivated polio vaccines (IPV ), Inactivated encephalitis B vaccine (JE), and inactivated hepatitis A vaccine (HAV). Methods for producing such vaccines are well known to those skilled in the art. For example, a method for producing an inactivated hepatitis A vaccine (HAV) using Vero cells is disclosed in Chinese Patent ZL 0210685.9.

The Vero cells used in the present invention can be prepared according to the method disclosed in the literature, for example, the method disclosed in Chinese Patent ZL 0210685.9; Alternatively, it can be readily obtained, for example, from ATCC (ATCC Series No: CCL-81). Kistner et al., Vaccine . 1998, 16: 960-968) or as disclosed in International Patent Publication WO 96/15231, the Vero cells are adapted to grow in serum-containing, serum-free or serum- and protein-free culture media. Cultivation of both Vero cells and reagents therefor is well known to those skilled in the art, as disclosed in U.S. Patent No. 4,783,407 and International Patent Publication No. WO 2003/049767. For growth in the serum-free culture medium, DMEM HAM's F12 minimal medium or other culture medium supplemented with organic salts, amino acids, sodium bicarbonate (2 g / L) and yeast or soy extract (1-10 g / L) Is used.

Bero cell destruction and dissolution :

The product of the berocellular lysing protein according to the present invention can be obtained by means of chemical (e.g. organic solvent), enzymatic (e.g. lysozyme and EDTA), mechanical or physical (e.g. homogenization, ultrasonic vibration, high-pressure homogenization, Lt; RTI ID = 0.0 > and / or < / RTI > Separation of intracellular products also includes a combination of mechanical and non-mechanical means. Preferred methods for performing cell destruction and lysis include chemical lysis, osmotic destruction, repeated freezing and thawing, enzymatic lysis of cells, ultrasound destruction and mechanical lysis of cells, and ultrasound destruction is more preferred.

Tablet :

A variety of methods can be used to purify the extracellular products (in the culture medium) or intracellular products (lysates) to facilitate product purification or removal of undesirable contaminants. One method is solid-liquid phase separation (e.g., centrifugation / precipitation, extraction, and filtration). Other methods are enrichment (e.g., evaporation, ultrafiltration, adsorption, and precipitation). Other methods are chromatography (e.g., molecular exclusion chromatography, ion exchange chromatography, chromatofocusing, hydrophobic chromatography, affinity chromatography, metal ion chelation chromatography, and covalent chromatography). These techniques can be easily performed by a person skilled in the art. If desired, the filtration, or may be used sterilization techniques, such as heating or irradiation (more information about the protein is purified, the literature [Ratledge C, Kristlansen B (2001 ) Basicbiotechnology, 2 nd ed. Cambridge University Press, Cambridge, UK].

In one embodiment, the purification includes pre-purification and micro-purification steps. Methods for pre-purification include isoelectric point precipitation, salt precipitation and organic solvent extraction, and organic solvent extraction is preferred. Microfiltering is accomplished by chromatography including molecular exclusion chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, metal ion chelation chromatography, and covalent chromatography.

In one embodiment of pre-purification, the pre-purification step comprises adding the cell solution obtained after cell destruction to an equal volume of trichloromethane, stirring the mixture for 20 to 30 minutes, mixing the mixture for 2 to 8 minutes Lt; 0 > C at 4000 rpm and taking the upper aqueous phase containing the protein.

In a preferred embodiment of the pre-purification step, the pre-purification step comprises adding the cell solution obtained after cell disruption to an equal volume of trichloromethane, stirring the mixture for 20 minutes, stirring the mixture for 20 minutes at 2-8 & centrifuging at rpm and taking the upper aqueous phase containing the protein.

In one embodiment of microfabrication, the microfiltering step comprises subjecting the sample obtained after pre-purification to microfiltration, hydrophobic chromatography purification and molecular exclusion chromatography and recovering the protein of interest of interest. The protein of interest has a collected protein peak in the same range as the collected peaks of the virus and its components in the vaccine production process. Non-limiting examples of such viruses and their components are hepatitis A virus and its components.

In a preferred embodiment, the hydrophobic chromatography tablet is a phenyl Sepharose 6FF hydrophobic tablet which is carried out as follows: After loading the sample at loading speed of 30-60 cm / h into loading buffer, 1.0 CV of 3 CVs (column volume) The column was washed with a mol / L PB solution (pH 6.0-7.5) and then linearly graded to a 0.02 mol / L PB solution (pH 6.0-7.5) of 5 CVs and finally eluted with a conductivity of less than 48 mS / cm Fractions are collected. The molecular exclusion chromatography is Sepharose 4 Fast Flow Molecule Exclusion Gel Chromatography, eluting with a 0.01 mol / L PBS (pH 7.4) solution at a flow rate of 45 cm / h and collecting a second elution peak .

In a preferred embodiment, the present invention provides a soluble protein of Vero cells prepared by the above method.

The soluble protein of Vero cells according to the present invention is a mixture of proteins having a molecular weight within the range of 11 KD, 17-34 KD, 55 KD, and 72 KD-170 KD.

The soluble protein of Vero cells according to the present invention is used to prepare a detection reagent for Vero cell HCPs. This protein can be used as an immunogen to establish a method for detection of Vero cell HCPs and as a standard for detection assays.

The soluble protein of Vero cells according to the present invention can be used as an antigen for producing a corresponding antibody. These antibodies, which are polyclonal antibodies, have the ability to specifically bind to soluble proteins of Vero cells. Non-limiting examples of antibodies are rabbit anti-lysis proteins of Vero cell IgG and guinea pig anti-lysis proteins of Vero cell IgG.

In another embodiment, the present invention provides a kit for the treatment and / or prophylaxis of a disease or condition associated with a disease or disorder, comprising a solid phase, a solid phase antibody, a ligand-conjugated antibody and a protein standard, Wherein the protein is a soluble protein of a cell.

In a preferred embodiment, the Vero cell HCPs assay kit according to the present invention further comprises a biotin-modified antibody and a ligand-conjugated avidin.

In another preferred embodiment, the Vero cell HCPs assay kit according to the present invention further comprises a 2,4-dinitrophenol modified antibody and a ligand-conjugated anti-2,4-dinitrophenol.

The assay kit according to the present invention may, if desired, comprise a reagent for detecting said ligand-conjugated (or labeled) avidin or ligand-conjugated (or labeled) anti-2,4-dinitrophenol.

Protein standards :

The soluble protein of Vero cells according to the present invention obtained according to the above production method is supplied as an assay kit according to the present invention as a protein standard for use in determining the residual content of Vero cell HCPs in a vaccine production and quality control process.

Antibodies :

As used herein, "antibody to soluble protein of Bero cell" refers to such an antibody produced using the soluble protein of Bero cell as an antigen. These antibodies, which are polyclonal antibodies, have the ability to specifically bind to soluble proteins of Vero cells. Non-limiting examples of antibodies are rabbit anti-lysis proteins of Vero cell IgG and guinea pig anti-lysis proteins of Vero cell IgG.

Antibodies against soluble proteins of Vero cells can be prepared by using soluble proteins of Vero cells as antigens using conventional methods known in the art to obtain corresponding antibodies.

For example, a rabbit anti-lysis protein of Vero cell IgG or a guinea pig anti-lysis protein of Vero cell IgG can be prepared by the following steps:

1) immunizing a rabbit or a guinea pig with a soluble protein of Vero cells according to the present invention four times, and then collecting blood, wherein:

For example, in the case of rabbits having a body weight of 2 kg, the primary immunization is performed at a dose of 0.5 mg protein / rabbit followed by a second immunization with a dose of 0.5 mg protein / rabbit after 4 weeks, The third immunization was performed after 10 days in the same manner, the fourth immunization was performed 2 weeks after the third immunization, and the blood was collected one week after the fourth immunization;

For example, in the case of guinea pigs having a body weight of 250 g, the primary immunization is performed at a dose of 0.2 mg protein / guinea pig, followed by a second immunization at a dose of 0.4 mg protein / guinea pig two weeks later , Followed by a third immunization in 10 days in the same manner, and finally a fourth immunization in 2 weeks after the third immunization, and then blood was collected one week later;

2) selecting antiserum having higher titer from each of the two animals and purifying the antiserum;

3) Detecting the potency after purification.

Purification of IgG can be performed by ammonium sulfate precipitation, caprylic acid-ammonium sulfate method, protein A affinity column chromatography and the like.

In the present invention, "titer" is expressed as the final titer, that is, the maximum dilution ratio of serum from which a positive antibody can be detected.

Ligand -conjugated antibody (or labeled antibody) :

As is well known to those skilled in the art, the purified antibody is labeled (or conjugated) with an enzyme (such as peroxidase, beta -D-galactosidase, alkaline phosphatase and glucose-6-phosphate dehydrogenase, etc.) ), Or labeled (or conjugated) with a fluorescent or radioactive isotope, which antibody is referred to as a "ligand-conjugated antibody" or "labeled antibody ". If the antibody is labeled with an enzyme, when the enzyme is reacted in the presence of a suitable substrate, the product of the enzyme reaction can be used as a label to qualitatively or quantitatively detect the antigen to which the antibody specifically binds. In addition, when a fluorescent substance-labeled or radioactive-isotope-labeled antibody is used, a fluorescent substance or radioactivity can be used as a label for qualitatively or quantitatively detecting an antigen to which the antibody specifically binds.

A "ligand-conjugated antibody" or "labeled antibody" further comprises an antibody modified with biotin, 2,4-dinitrophenol or the like. Biotin is specifically bound by avidin, while 2,4-dinitrophenol is specifically bound by anti-2,4-dinitrophenol. Thus, the labeled antibody can be detected qualitatively or quantitatively using enzyme-, nuclide-, or fluorescein-labeled avidin or anti-2,4-dinitrophenol.

The labeling antibody can be prepared by reacting a biotin-labeled reagent (NHS-LC-biotin, Pierce Co., Ltd) and / or a peroxidase containing a coupling agent (maleimide activated HRP, Pierce Co., have.

In some embodiments of the Vero cell HCPs assay kit according to the present invention, the label in the ligand-conjugated antibody includes, but is not limited to, enzymes, nuclides and fluorescein. The enzyme may be selected from, without limitation, peroxidase,? -D-galactosidase, alkaline phosphatase, and glucose-6-phosphate dehydrogenase; The nuclide is selected from ³ H, ¹⁸⁸ Re, ¹³¹ I, without limitation, and the fluorescein is selected from the group consisting of fluorene isothiocyanate, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, europium ( 3, such as ^{3 +} Eu), terbium (Tb ^{+ 3),} and cerium (Ce ^{+ 3)} are selected from lanthanide chelates.

When the label is an enzyme, the kit according to the present invention further comprises auxiliary reagents, including a substrate solution, a dye solution, a reaction stop solution and a wash buffer solution for an enzyme reaction.

An illustrative auxiliary reagent has the following composition:

1. Substrate solution: 3% H ₂ O ₂ prepared in phosphate-citrate buffer (pH 5.0);

2. Dye solution: 0.0004 mol / L TMB in methanol or 0.0013 mol / L TMB in hydrochloride solution;

3. Stopping solution: 2 mol / L sulfuric acid;

4. Wash buffer solution: PBS solution.

Solid phase :

As used herein, the term "solid phase" refers to any solid, such as an ELISA assay plate, a protein chip carrier, such as a membrane, a glass slide, etc., in which multiple liquid samples can be analyzed individually using the method according to the present invention Lt; / RTI > As used herein, a solid "reaction well" refers to a defined region on a solid phase that is provided to a sample receiving region. Typical solid phase reaction wells are obtained by forming indentations on the surface of the plate, each recessed area containing a volume of sample and volume of buffer or wash solution added at any stage of the crystallization process Is sufficient. As used herein, "measurement" of a target molecule refers to determining, quantifying, or determining the content of the analyte or target molecule.

The Vero cell HCPs assay kit provided by the present invention can be effectively used to detect the residual content of Vero cell HCPs according to the following steps: collecting a sample; Performing the crystal using the Vero cell HCPs assay kit; And analyzing the results.

Determination means :

Antibodies are key reagents in many of the techniques used in medicine, veterinary medicine and other fields. Such determination techniques include many commonly used immunological assays such as protein chips, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC) Analysis techniques. For a general introduction to these techniques, see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1987. Additionally, immunoassay provides a means for immunohistochemistry (IHC) staining or visualization of the immunofluorescent (IF) in tissue samples. See, Principles and Practice of Immunoassay, Christopher P. Priceand David J. Neoman, Stockton Press, New York, N.Y., 1991.

In a particular embodiment, the analytical means in the present invention is that the polyclonal antibody to the specific molecular form of the bero cytolytic protein to be determined is a total (noncomplexed + antibody-complexed) or glass (noncomplexed) It is an immunoenzymometric analysis used to detect the content of BERRO cell lysate protein. For example, a non-limiting example of an immunoenzymometric assay is performed as follows:

1. Plate Coating: The surface of the reaction well of the assay plate is coated with an antibody against the soluble protein of the optimal concentration of Vero cells. Optimal antibody concentration is determined from the standard curve plotted using a known concentration of Bero cytolytic protein, which has the required sensitivity and accuracy within the essential range of effective concentrations. For Vero cell lysate proteins, the effective concentration range of Vero cell lysate protein that can be detected by the assay kit according to the present invention is 62.5 ng / ml to 4000 ng / ml. One of ordinary skill in the art will readily determine whether adequate sensitivity and accuracy are achieved within the essential range, without undue experimentation.

2. Wash plate: discard along with the coating solution, add wash buffer (approx. 400 μL / well) and discard. This cleaning step is repeated as many times as necessary. The wash buffer may be 0.01 mol / L phosphate buffer (containing 0.0027 mol / L potassium chloride, 0.137 mol / L sodium chloride, pH 7.4, 0.01% w / v Triton X-100).

3. Plate blocking: Blocking buffer (1% BSA / coating buffer containing 0.1% Triton X-100) containing protein and surfactant is added to the reaction well. This well can be kept shut.

4. Add samples and standards: Plates are washed as described above. 100 μL each of the standard and the determined sample is added to the individual reaction wells of the plate, and then 50 μL of the reagent is added to each well. The mixture was lightly mixed for 15 seconds and then incubated at 37 DEG C for 60 minutes. The reaction solution was removed, and the reaction plate was washed five times with buffer. After removing excess water, 50 μL of a dye solution was added to each well and incubated at 37 ° C for 15 minutes, at which point the reaction was stopped. The reaction plate was placed on an ELISA reader and the optical density was read. The different dye reactions in each well represent different values of optical density.

5. Examples of suitable enzyme substrates useful for quantitating the color of the complex are: nitrophenyl phosphate for alkaline phosphatase or tetramethylbenzidine sulfonate (TMBS) for horseradish peroxidase. The degree of color development can be read as the absorbance unit (AU; for p-nitrophenol, the absorbance is read at 405 nm, while for TMBS, the absorbance is read at 450 nm), which is the index of the HCP content in the determined sample . The exact concentration of HCP can be determined by reading the absorbance of the determined sample and referring to a standard curve prepared using the HCP standard.

6. If a sufficient detection signal is obtained during the reading process, the signal is measured using a full-wavelength ELISA reader or a fluorescent ELISA reader or the like.

7. Data processing is performed by creating calibration curves from the detection signals obtained using standard solutions of Vero cell lysate proteins of known concentrations. The calibration curve is used for the calculation to obtain the concentration of Vero cell lysing protein in the determined sample.

Beneficial effect

The present invention provides a soluble protein of Vero cells and a process for producing the same. This protein can be used as an immunogen to establish the determination method for Vero cell HCPs and as a standard for detection assays. The soluble protein of Vero cells according to the present invention has a very wide application range. They not only can be used for the production of intracellular non-secretory virus vaccines against viruses such as hepatitis A virus, but also have a high specificity, high sensitivity and excellent reproducibility, Can be used for analysis.

The present invention also provides a corresponding Vero cell HCPs assay kit with strong specificity, high sensitivity and excellent reproducibility. This can be used for quality control and analysis in the production of other vaccines produced from Vero cells as well as for the production of non-secreted virus vaccines against viruses such as hepatitis A virus.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a flow chart of a method for producing a Vero cell soluble protein.
Figure 2 shows the chromatogram of Bero cell purification on a Phenyl Sepharose 6 high-speed flow hydrophobic gel.
Figure 3 shows the chromatogram of Bero cell purification on a Sepharose 4 fast flow molecular exclusion gel.
Figure 4 shows the staining pattern of SDS-PAGE of the standard product of Vero cell lysate protein.
Figure 5 shows a standard curve prepared using a sandwich ELISA for a standard of Vero cell lysate protein.
FIG. 6 shows the specificity test of the method for determining Bero-cell HCPs according to the present invention.

The present invention will be described in more detail with reference to the following specific examples. These examples are merely illustrative of the invention, not limiting the scope of the invention. In the following examples in which no particular conditions are specified, the experimental procedure is generally carried out according to the usual conditions or conditions recommended by the manufacturer. Ratios and percentages are by weight or volume unless otherwise indicated.

Source of material:

Inoculation cells of Vero cells: ATCC series No: CCL-81

Experimental animal

Rabbits (normal grade, weight: 2 kg), obtained from Shanghai Puxin Biological Co., Ltd, Shanghai, China;

Guinea pig (clean grade, weight: 250 g), obtained from the Shanghai Laboratory Animal Center (Chinese Academy of Science).

Key equipment and reagents:

Ultrasonic cell shredder: SONICS VCF1500, maximum output power 1500 W; Ultrafilter: Pellicon 2 Cassette Filter; Biomax-100A (MWCO = 100 KD) (Millipore Corp., Millipore Corp.); (GE Healthcare Co., Ltd), Chromatography column: BPG140 / 950; INdEX140 / 500 (manufactured by GE Healthcare Corporation, Elsevier); ultrafiltration concentrator (MWCO = 100 KD) (Millipore Corporation; Sepharose 4 Fast Flow Type Molecular Exclusion Gel (Gly Healthcare Corporation); Protein-A Affinity Column, Hose < RTI ID = 0.0 > ELISA plate (Costar Co., Ltd); ELISA reader, Thermo Multiskan MK 3 type (Thermo Multiscan MK 3 type) ; Bovine serum, purchased from Lanzhou Minhai Biotechnology Co., Ltd, Gansu, China, batchno .: 051203; cell culture solution: 199 medium (GIBCO); bovine serum (BSA, fraction V), which was prepared from Calbiochem Co., Ltd and was packaged by Yicheng Bioscience Co., Ltd, Shanghai, China; chlorine anti- Rabbit-HRP, goat anti-guinea pig-HRP, all purchased from KPL Co., Ltd. egg white albumin (albumin from egg white), manufactured by Sigma Corporation; hepatitis A (HAV), hepatitis A virus strain pHM 175, 10.81 ug / ml, purchased from BIODESIGN International; anti-HAV IgG standard, 75 IU / ml, National Institute for Fertility Controls, Purchased from the National Institute of Pharmaceutical and Biological Products, China; Trypsin, manufactured by BD Co., Ltd; Lactoalbumin hydrolyzate, manufactured by GIBCO Co., Ltd; Proclin 300, manufactured by Supelco Co., Ltd; TMB hydrochloride, purchased from Suzhou BEC Biological Technology Co., Ltd, Jiangsu, China.

Example  1: Preparation of Vero cell soluble protein

Manufacturing method:

1. Cell culture and harvest: Cells obtained from the Bero cell strain of a tube obtained from the working cell bank were inoculated into a cell culture bottle, and 199 medium containing 10% bovine serum was added, followed by incubation at 37 ± 1 ° C Lt; / RTI > The resuspended cells were grown in a dense monolayer, and the obtained Vero cells were digested with trypsin for passage and proliferation, incubated at 37 占 1 占 for 25 days, and then harvested on the 25th day. Cells were digested with trypsin and centrifuged at 3500 rpm for 30 min at 4 ° C. Cell pellets were collected for later use as a source for preparing samples of soluble proteins.

2. Cell destruction and lysis: The harvested Vero cells were disrupted and lysed by ultrasonication at an output of 1400 W until no intact cell structure was present.

3. Extraction of Protein Using Chloroform: The same volume of chloroform was added to the destroyed cell solution, and the mixture was stirred on a reciprocating shaker at 300 rpm for 20 minutes to make the chloroform completely in contact with the cells. After allowing the cell debris to settle, the mixture was centrifuged at 4000 rpm for 20 minutes at 4 ° C, and the upper aqueous phase containing the protein was removed. The same amount of 0.01 mol / LPBS (pH 7.8) was added to the centrifuge cup and extraction was repeated 4 times.

4. Concentration: All extraction solutions were combined and concentrated using MWCO 100 KD ultrafiltration membrane. PB buffer salt was added to the ultrafiltration concentrate and completely dissolved to obtain a PB final concentration of 1.0 mol / L (pH 6.8).

5. Purification: The concentrate was adsorbed onto a Phenyl Sepharose 6 high-speed flow type hydrophobic gel at a flow rate of 50 cm / h and washed with 3 CVs of 1.0 mol / LPB (pH 6.8) solution after adsorption was terminated and 5 And linearly eluted with 0.02 mol / LPB (pH 6.8) solution of CVs. Eluted fractions having a conductivity of less than 48 mS / cm were collected and concentrated using an MWCO 100 KD ultrafiltration membrane. The resulting concentrate was loaded onto a Sepharose 4 high-speed flow-type molecular exclusion gel and eluted at a flow rate of 45 cm / h using 0.01 mol / LPBS (pH 7.4) solution, and then a second elution peak was collected.

6. Additional concentration: The collected solution was first concentrated to 200 mL using MWCO 100 KD ultrafiltration membrane and then concentrated to 30 mL using a 50 mL ultrafiltration centrifuge tube (MWCO = 100 KD) (A standard product of Vero cell lysate protein or Vero cell HCPs).

Experimental Results: The concentration of the bell-cell lysis protein was determined to be 540 μg / mL by the Lowry method. The relevant experimental data of the present invention are shown in Table 1 below.

Changes in the protein content of the sample at different purification steps Purification step Bero cell destruction Protein extraction concentration Hydrophobic tablets Molecular exclusion chromatography Volume (mL) 2300 10600 2250 12286 2543 Protein content (μg / mL) 17690 2137 3779 192 16 BER cell protein removal rate - 44.3% 79.1% 94.2% 99.9%

The results of SDS-PAGE electrophoresis and silver staining are shown in Figure 4, where: 1 : standard molecular weight protein; 2 ; The soluble protein of the uncleaved Bero cells obtained in step 4; 3 : Is a soluble protein of Vero cells. The results indicate that the soluble protein of the Vero cells obtained after purification contains various protein components within the molecular weight range of 55 KD, 11 KD, 170 to 72 KD and 34 to 17 KD.

Example  2. Preparation of Antibodies to Soluble Proteins of Vero Cells

1. Immunization of rabbits:

Three rabbits were immunized. Primary immunizations were performed subcutaneously at 6-8 sites on the back of each rabbit using an emulsion of 0.5 mg of Vero cell lysate protein as prepared in Example 1, which was completely emulsified in the same volume of complete Freund's adjuvant. Emulsion. After 4 weeks, the secondary immunization was subcutaneously and intramuscularly emulsified in multiple sites using an emulsion of 0.5 mg of Vero cell lysate protein as prepared in Example 1, which was completely emulsified in the same volume of incomplete Freund ' s adjuvant. . After the second immunization and 10 days later, the third immunization was performed in the same manner. Finally, after the third immunization, two weeks later, the fourth immunization was performed. Four weeks after immunization, blood was collected and analyzed for activity.

Serum Potency of Immunized Rabbits rabbit Serum volume (mL) Potency One 35 1: 106 2 40 1: 106 3 41 1: 106

2. Immunization of guinea pigs:

Three guinea pigs were immunized. Primary immunizations were performed subcutaneously at 5 to 6 sites on the back of each guinea peak using an emulsion of 0.2 mg of Vero cell lysate protein as prepared in Example 1, which was completely emulsified in the same volume of complete Freund's adjuvant. This emulsion was performed by injection, and each area was injected with 0.1 ml of emulsion. After two weeks, the secondary immunization was performed by injecting the subcutaneous emulsion into multiple sites using an emulsion of 0.4 mg of Vero cell lysate protein as prepared in Example 1, which was completely emulsified in the same volume of incomplete Freund's adjuvant Respectively. After the second immunization and 10 days later, the third immunization was performed in the same manner. Finally, after the third immunization, two weeks later, the fourth immunization was performed. Four weeks after immunization, one week later, blood was collected and the titer was measured.

Serotypes of immunized inoculated guinea pigs Guinea pig Serum volume (mL) Potency One 5 1: 105 2 4 1: 105 3 5.5 1: 105

3. Purification of antiserum:

Antiserum with high titer was individually selected from each of the two animals for purification. Affinity chromatography was performed using a commercially available pre-packaged protein-A Sepharose affinity column to obtain an anti-lytic protein of Vero cell IgG. Absorbance was determined at 280 nm and 260 nm and the concentration of antibody protein was calculated using the following equation:

[Mathematical Expression]

C (mg / ml) = 1.45 x OD _280nm - 0.74 x OD _{260 nm}

Concentration of antibody protein after purification OD _{280 nm} OD _{26 nm} Dilution rate Concentration of antibody protein (mg / mL) 1 # guinea pig antibody 0.226 0.163 20 4.109 2 # guinea pig antibody 0.208 0.154 20 3.722 3 # guinea pig antibody 0.196 0.149 20 3.449 1 # rabbit antibody 0.204 0.148 20 3.696 2 # rabbit antibody 0.215 0.156 20 3.895 3 # Rabbit antibody 0.182 0.141 20 3.163

4. Determination of antibody titer after purification:

Antibody titers were determined by indirect ELISA. Specifically, the soluble protein of Vero cells as prepared in Example 1 was diluted to 10 μg / mL using carbonate buffer (0.05 mol / L CB, pH 9.6). The microtiter plate was coated with 100 [mu] L of diluent per well and incubated overnight at 4 [deg.] C. The next day, the plates were washed twice with PBS. Thereafter, the plate was blocked with 125 [mu] L / well of 2% BSA, incubated at room temperature for 2 hours, and then washed twice with PBS. IgGs for lysed proteins of Vero cells obtained in step 3 were diluted to 1 mg / mL, respectively, and then 10-fold serial dilutions were added to the microtiter plates at 100 μL / well. The plate was incubated at 37 DEG C for 60 minutes, washed and then patted dry. Thereafter, 100 μL of enzyme-conjugated secondary antibody (goat anti-rabbit-HRP or goat anti-guinea pig-HRP) was added to each well, and the plate was incubated at 37 ° C. for 30 minutes and then washed and patted . Dye reagent A and Dye reagent B solutions were added to each well, and the plate was incubated at 37 占 폚 for 15 minutes. 2 mol / L sulfuric acid was added to stop the reaction. Colorimetry was determined on a microtiter reader and antibody titers were calculated.

Antibody titer after purification 1 # guinea pig antibody 2 # guinea pig antibody 3 # guinea pig antibody 1 # rabbit antibody 2 # rabbit antibody 3 # Rabbit antibody Potency 1: 106 1: 105 1: 105 1: 105 1: 106 1: 105

Example  3. Preparation of enzyme-conjugated antibodies

IgGs (1 # guinea pig IgG for the lysed protein of Vero cells and 2 # rabbit IgG for the lysed protein of Vero cells) were separately selected for the lysed protein of Vero cells obtained in step 3 having the highest titer, And labeled with horseradish peroxidase (HRP) using the sodium periodate method (Luo Jiali et al., Acta Biochimica meat Biophysica Sinica , 1981, 13: 1).

The anti-lysis protein of HRP-Vero cell IgG Enzyme-conjugated antibody OD _{403 nm} OD _{280 nm} Concentration of HRP (mg / mL) Concentration of IgG (mg / mL) Marking rate (LR) Mole ratio HRP-rabbit anti-lysis protein of Vero cell IgG 1.39 2.60 0.56 1.35 0.53 1.66 HRP-guinea pig anti-lysis protein of Vero cell IgG 1.20 2.80 0.48 1.51 0.42 1.27

Example  4. Vero cells IgG of Guinea Pig  Determination of optimal coating concentration of anti-lytic protein

The guinea pig anti-lysed protein of Vero cell IgG as prepared in Example 2 was diluted to 5-20 μg / mL using 0.05 mol / L CB (pH 9.6). The microtiter plate was coated with a dilution at a concentration of 100 [mu] L / well and incubated overnight at 4 [deg.] C. The next day, the plates were washed twice with 0.01 mol / LPBS (pH 7.4). Thereafter, 150 [mu] L per well of 2% BSA-PBS was added to the plate, blocked for 2 hours at room temperature, then washed twice with PBS and air-dried. Continuous dilutions of the soluble protein standards of Vero cells (as prepared in Example 1, the concentrations set forth in Table 7) were added to the plates and allowed to react for 60 minutes at 37 ° C. Thereafter, the plate was washed four times and gently patted to dry. Enzyme-conjugated antibody HRP-rabbit anti-lysed protein of Vero cell IgG was diluted (1: 500-1: 1000) with PBS-T (containing 10% calf serum and 0.03% Prochlor 300). To this plate was added 100 μL of dilution per well and allowed to react for 60 minutes at 100 ° C., followed by washing with PBS-T four times, followed by gentle tapping. Thereafter, the color development reaction was allowed to proceed for 10 minutes at 37 DEG C, and then the reaction was stopped and color development was determined. The results indicated that the optimal coating concentration for the plate was in the range of 5-20 μg / mL.

Optimization of coating concentration of guinea pig anti-soluble protein of Vero cell IgG antibody OD value (zero blank) Coating concentration Standard product 5 μg 10 μg 20 μg 4000 ng 2.495 2.753 3.011 2000 ng 1.267 1.382 1.690 1000 ng 0.480 0.599 0.718 500 ng 0.320 0.373 0.387 250 ng 0.152 0.178 0.225 125 ng 0.065 0.087 0.105 62.5 ng 0.030 0.027 0.037 Regression equation y = 1.5852x + 44.474
R ² = 0.9963 y = 1.449x + 15.935
R ² = 0.9985 y = 1.3054x - 17.45
R ² = 0.9958

Example  5: Vero cells IgG of HRP - Determination of optimal dilution ratio of rabbit anti-lysing protein

(10 μg / mL) coated with purified guinea pig anti-lysis protein of Vero cell IgG as prepared in Example 2 was added to a serial dilution of the standard of Vero cell lysate protein (as prepared in Example 1 , The same concentration as described in Table 8) was added and reacted at 37 캜 for 60 minutes, followed by washing 4 times, followed by tapping and drying. Enzyme-conjugated antibody HRP-rabbit anti-lysed protein of Vero cell IgG was diluted (1: 500-1: 1000) with PBS-T (containing 10% bovine serum and 0.03% Proclean 300) . 100 [mu] L of diluted enzyme-conjugated antibody was added to each well of the plate and mixed well by stirring. The plate was incubated at 37 ° C for 60 minutes, then washed 4 times with PBS-T and gently patted to dryness. Thereafter, the color development reaction was allowed to proceed at 37 DEG C for 10 minutes, and then the reaction was stopped and the degree of color development was determined. The results showed that the optimal dilution ratio for the HRP-rabbit anti-lysis protein of Vero cell IgG ranged from 1: 500 to 1: 1000.

The dilution ratio of HRP-rabbit anti-lytic protein of Vero cell IgG OD value (zero blank) The dilution ratio of Vero cell IgG to HRP-rabbit anti-lysis protein Standard product 1: 500 1: 750 1: 1000 4000 ng 3.182 2.913 2.884 2000 ng 1.635 1.481 1.411 1000 ng 0.728 0.693 0.567 500 ng 0.441 0.408 0.349 250 ng 0.203 0.194 0.183 125 ng 0.100 0.094 0.079 62.5 ng 0.064 0.031 0.037 Regression equation y = 1.2556x - 5.9
R ² = 0.9989 y = 1.3723x - 6.4208
R ² = 0.9994 y = 1.38x + 47.379
R ² = 0.9971

Example  6: Dual antibody sandwich ELISA Establishment of

Plate Coating: Purified guinea pig anti-lysing protein of Vero cell IgG was diluted to 5-20 μg / mL using 0.05 mol / L CB (pH 9.6). The microtiter plate was coated with a dilution at a concentration of 100 [mu] L / well and incubated overnight at 4 [deg.] C. The next day, the plates were washed twice with 0.01 mol / LPBS (pH 7.4). Then, 150 [mu] L per well of 2% BSA-PBS was added to the plate, blocked at room temperature for 2 hours, then washed twice with PBS and air-dried. The plate was sealed in a ziplock bag with a desiccant on the small package, and stored at 4 [deg.] C for subsequent use.

Sample addition: A standard solution of Vero cell lysate protein as prepared in Example 1 was diluted to 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, / mL and 62.5 ng / mL, respectively. 100 μL of each of the dilutions of the standard was added to each well of the plate with 0.01 mol / L PBS-T and the sample to be detected. After allowing the mixture to react for 60 minutes at 37 ° C, the plate was washed 4 times with PBS-T and gently patted to dry.

Addition of HRP-rabbit anti-lysis protein of Vero cell IgG: The HRP-rabbit anti-lysate protein of Vero cell IgG as prepared in Example 2 was dissolved in PBS-T (containing 10% bovine serum and 0.03% ) At a suitable dilution ratio (1: 500-1: 1000). 100 [mu] L of diluted Vero cell IgG HRP-rabbit anti-lysis protein was added to each well of the plate and mixed well by stirring. After allowing the plate to react for 60 minutes at 37 ° C, the plate was washed 4 times with PBS-T and gently patted to dry.

Color development: To each well was added substrate dye reagent A (0.1 mol / L sodium acetate-citrate buffer solution, 0.0038 mol / L hydrogensperoxidase, 20000 units / L gentamycin, pH 5.0) and coloring reagent B (0.02 mol / L tris-HCl buffer, 0.0005 mol / L disodium ethylenediamine tetraacetic acid dihydrate, containing 0.0013 mol / L TMB hydrochloride) was added and the mixture was allowed to react at 37 占 폚 for 10 minutes.

Determination of color: 50 μL of stop solution (2 mol / LH ₂ SO ₄ ) was added to each well and mixed well by stirring. The OD values of each well were read on an ELISA reader at 450 nm.

Calculation: A standard curve was prepared by subtracting the OD value of the standard (0, ng / mL sample dilution solution: 0.01 mol / L PBS-T) from the OD values of the standard products of different concentrations and the sample (zero blank). Linear regression equations and R ² values. The content of Vero cell HCPs in the sample was calculated based on the standard curve.

Example  7: Correlation of the standard curve

A standard of Vero cell lysate protein as prepared in Example 1 was diluted to a concentration of 4000 ng / mL to 62.5 ng / mL. Different concentrations of dilutions were detected as described in Example 6. The curve shows the OD value of the standard product (i.e., sample dilution solution: 0.01 mol / L PBS-T) from the OD value of the standard product (zero blank) on the vertical axis representing the concentration of the standard product of Vero cell lysate protein and 0 ng / Plotted on the abscissa representing the 1000 x OD value obtained by subtracting < RTI ID = 0.0 > a < / RTI > Curve correlations appear when the concentration of the standard is within the range of 62.5 to 4000 ng / mL (R ² ≥ 0.99).

The result of determination of standard product of Vero cell soluble protein The content (ng / mL) of the standard product of Vero cell lysate protein 62.5 125 250 500 1000 2000 4000 OD _450nm 0.057 0.100 0.217 0.396 0.880 1.623 3.135

Example  8: Vero cells for determination HCPs  analysis Kit

A reaction plate coated with guinea pig anti-lysis protein of Vero cell IgG

96 well x 1 plate

The standard (dissolved protein of purified BERO cell, 8000 ng / mL as in Example 1)

500 μL × 1 bottle

HRP-rabbit anti-lysis protein of BERO cell IgG 50 μL × 1 bottle

Enzyme dilution buffer 20 ml × 1 bottle

20 x PBS wash buffer 50 ml x 1 bottle

Pigment reagent A 7 ml × 1 bottle

Pigment reagent B 7 ml × 1 bottle

Stop solution (2 mol / LH ₂ SO ₄ ) 7 ml × 1 bottle

Example  9: Vero cells HCPs  analysis Kit

A reaction plate coated with rabbit anti-lysis protein of Vero cell IgG

96 well x 1 plate

The standard (dissolved protein of purified BERO cell, 8000 ng / mL as in Example 1)

500 μL × 1 bottle

HRP-rabbit anti-lysis protein of BERO cell IgG 50 μL × 1 bottle

Enzyme dilution buffer 20 ml × 1 bottle

20 x PBS wash buffer 50 ml x 1 bottle

Pigment reagent A 7 ml × 1 bottle

Pigment reagent B 7 ml × 1 bottle

Stop solution (2 mol / LH ₂ SO ₄ ) 7 ml × 1 bottle

Example  10: Vero cells HCPs  analysis Of kit  Specificity test

An assay kit as described in Example 8 is first used to detect the amount of host cell protein in the intermediate product during the production of the Vero cell vaccine. Such an intermediate may comprise components other than the host cell protein. In particular, these components in particular, calf serum, the cell culture solution, BSA, ovalbumin, hepatitis A virus (HAV), wherein -HAV IgG, trypsin and Lactobacillus, including albumen hydrolyzate will not interfere with the determination according to the kit Should not. HAV (1: 4 dilution, 2.7 [mu] g / mL), anti-HAV (1: 4 dilution, according to the IgG (2 IU / mL), 0.25% trypsin and 2% lactose sample described in example 6 using the albumin hydrolysates as can be determined it is carried out to demonstrate the specificity of the determination method.

The results are shown in Figure 6, where: 1) is a standard product of Vero cell lysate protein; 2) is PBS-T; 3) is a cell culture medium; 4) is calf serum; 5) is BSA; 6) is egg white albumin; 7) is HAV; 8) is HAV-IgG; 9) is trypsin; 10) is a lactoalcohol hydrolyzate. The results show that the assay kits for crystals according to the present invention do not cause cross-reactions with calf serum, cell culture solutions, BSA, albumin albumin, HAV, anti-HAV IgG, trypsin and lactoalcohol hydrolysates and are therefore very specific .

Example  11: Vero cells HCPs  analysis Of kit  Accuracy check

Using an assay kit as described in Example 8, OD _450nm values of 10 wells containing 1000 ng / mL of the standard product on the same plate were determined according to the method described in Example 6. This determination was repeated three times and the intra-batch coefficient of variation (CV%) was calculated to be 6.0%.

Determined OD _{450 nm} of 1000 ng / mL standard Decision number One 2 3 4 5 6 7 8 9 10 CV% OD _450nm 0.730 0.671 0.662 0.625 0.631 0.610 0.619 0.623 0.719 0.652 6.2% 0.735 0.711 0.660 0.637 0.635 0.622 0.620 0.647 0.722 0.664 6.4 & 0.834 0.774 0.758 0.713 0.693 0.773 0.710 0.737 0.782 0.768 5.5%

Example  12: Determination and determination of reproducibility of vaccine samples

Four batch samples of the inactivated hepatitis A vaccine were prepared according to the description of Chinese patent ZL 0210685.9. These samples were detected six times according to the method described in Example 6 using an assay kit for crystals as described in Example 8. [ The results of the content of Vero cell HCPs are shown in Table 11, with an average CV of 6.2%, which shows excellent reproducibility.

Concentration of Vero cell HCPs in vaccine samples

Regression equation of standard product The content of Vero cell HCPs (ng / mL) Placement of vaccine samples 1 Placement of vaccine samples 2 Placement of vaccine samples 3 Placement of vaccine samples 4 Experiment 1 y = 1.063x + 10.497
R ² = 0.999 381 401 328 223 Experiment 2 y = 1.0665x + 9.0876
R ² = 0.9986 380 342 292 242 Experiment 3 y = 1.2742x-32.471
R ² = 0.999 364 371 262 221 Experiment 4 y = 1.6439x-5.9769
R ² = 0.9976 376 415 284 224 Experiment 5 y = 1.3581x-38.712
R ² = 0.9951 389 427 292 250 Experiment 6 y = 1.2999x-11.772
R ² = 0.9988 403 419 277 246 Mean (ng / mL) 382 395 289 234 CV (%) 3.4 8.3 7.7 5.5

Example  13: Total protein in the vaccine sample decision And Vero cells HCPs of decision Comparison of

The total protein in the vaccine samples of the four place determined by the Lowry method, and compared the results to determine a result of the fire within the Vero cell HCPs active hepatitis A vaccine produced from Vero cells. This demonstrates that the present invention provided as a means for quality control and management of inactivated hepatitis A vaccine produced from Vero cells is target-oriented. The quality standard of the vaccine samples increases accordingly, thus ensuring a higher level of vaccine stability.

Comparison of results of two detection methods Placement of vaccine samples 1 Placement of vaccine samples 2 Placement of vaccine samples 3 Placement of vaccine samples 4 Total protein
(μg / mL) 3.3 2.4 2.2 1.5 Vero cell HCPs (ng / mL) 382 395 289 234

Example  14: Recovery rate check

Using an assay kit as described in Example 8 and following the steps of Example 6, dilutions of HCP standards diluted in batch 3 vaccine samples (after double dilution) and dilutions of HCP standards diluted in PBS-T Respectively. The recovery of HCP in the vaccine samples averaged 96% (see Tables 13 and 14), indicating that the specificity of the method for detecting HCPs according to the present invention was not affected by the substrate of the vaccine sample.

Determination results of standard (HCPs) and vaccine samples (double diluted) of Vero cell lysate protein The content of the standards of HCPs (ng / mL) 62.5 125 250 500 1000 2000 4000 Vaccine samples (ng / mL) 133 OD _450nm 0.057 0.100 0.217 0.396 0.880 1.623 3.135 Vaccine sample OD _450nm 0.130

Recovery rate check The standard (133 ng / mL) diluted with the vaccine sample OD value 0.261 0.311 0.401 0.578 0.940 1.664 3.214 The results of detection of HCPs content (ng / mL) 183 246 361 586 1048 2088 3946 The theoretical content of HCPs (ng / mL) 194 255 378 624 1116 2099 4066 Recovery rate (%) 94.3 96.7 95.6 93.9 93.9 99.5 97.0

The present invention is not intended to be limited by the specific embodiments described herein, which are intended as single examples of individual aspects of the invention. Functionally equivalent methods and components are included within the scope of the present invention. Indeed, various modifications of the invention in addition to those disclosed and described herein will become apparent to those skilled in the art from the foregoing description and the appended claims. Such modifications are intended to fall within the scope of the appended claims. The description of each reference mentioned above is incorporated herein by reference in its entirety.

Claims (24)

a) culturing and harvesting Vero cells;
b) destroying and dissolving Vero cells;
c) pre-purifying the lysed protein of Vero cells;
d) Concentrating the obtained product after pre-purification with a MWCO = 100 KD microfiltration membrane to obtain a concentrate; And
e) loading the concentrate into loading buffer at a flow rate of 30-60 cm / h onto a Phenyl Sepharose 6FF hydrophobic gel, then washing the column with 3 CVs of 1.0 mol / L pH 6.0-7.5 PB solution, 5 CVs of 0.02 mol / L pH 6.0-7.5 PB solution, finally eluting fractions having a conductivity of less than 48 mS / cm were collected and concentrated using a MWCO = 100 KD microfiltration membrane to give a second concentration Water,
The obtained second concentrate was loaded on a Sepharose 4 high-flow-type molecular exclusion gel, eluted at a flow rate of 45 cm / h using 0.01 mol / L pH 7.4 PB solution, and then the second elution peak was collected Collecting a solution comprising the desired protein of interest having a collected protein peak in the same range as the collected peak of the virus and its components in the course of production of the vaccine,
Concentrating the collected solution using a MWCO = 100 KD ultrafiltration membrane, and MWCO = 100 KD tube to obtain a soluble protein of Vero cells. The method of claim 1, wherein said cell destruction and lysis comprises chemical cell lysis, osmotic destruction, repeated freezing and thawing, enzymatic cell lysis, ultrasound destruction and mechanical cell lysis. 3. The method of claim 2, wherein said cell destruction and lysis is by ultrasound destruction. The method according to claim 1, wherein the pre-purification method is an organic solvent extraction method. 5. The method of claim 4, wherein the organic solvent extraction comprises adding the cell solution after destruction to an equal volume of trichloromethane, stirring the mixture for 20-30 minutes, mixing the mixture at 2-8 [deg.] C at 4000 rpm ≪ / RTI > and a step of taking an upper aqueous phase containing the protein. 2. The method of claim 1, wherein the virus and its components are hepatitis A virus and its components. A process for the preparation of a compound of formula I, which is prepared using the process of any one of claims 1 to 6,
A mixture of soluble proteins of Vero cells, a mixture of proteins having molecular weights ranging from 11 KD, 17-34 KD, 55 KD, and 72 KD-170 KD. delete An antibody to a mixture of lysed proteins of Vero cells according to claim 7. 10. The antibody of claim 9, which is an anti-lysis protein antibody produced in guinea pigs or rabbits and having an IgG of Vero cells. delete A solid phase, a solid phase antibody, a ligand-conjugated antibody and a protein standard, wherein the antibody is an antibody against a mixture of soluble proteins of Vero cells, the protein standard is a soluble protein of Vero cells, Wherein the mixture is prepared using the method of any one of claims 1-6. 13. The kit according to claim 12, further comprising a biotin-modified antibody and a ligand-conjugated avidin. 13. The kit according to claim 12, further comprising an antibody modified with 2,4-dinitrophenol and a ligand-conjugated anti-2,4-dinitrophenol. 13. The kit according to claim 12, wherein the antibody against the soluble protein of the Vero cell is an anti-soluble protein antibody produced in guinea pig or rabbit and having IgG of Vero cell. 13. The kit according to claim 12, wherein the label in the ligand-conjugated antibody is one of enzymes, nuclides or fluorescein. 17. The kit according to claim 16, wherein the enzyme is one of peroxidase,? -D-galactosidase, alkaline phosphatase or glucose-6-phosphate dehydrogenase. 17. The kit according to claim 16, wherein the nuclide is selected from one of ³ H, ¹⁸⁸ Re, and ¹³¹ I. 17. The method according to claim 16, wherein the fluorescein is selected from one of fluorene isothiocyanate, tetraethylodamine, tetramethylrhodamine isothiocyanate or trivalent lanthanide chelate. Analysis Kit. 13. The kit according to claim 12, wherein the solid phase is an ELISA assay plate or a protein chip carrier.
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