SEC-HPLC-based method for quantitatively detecting VP number of adenovirus

Abstract

The invention relates to the technical field of biological detection, in particular to a method for quantitatively detecting the number of adenovirus VP based on SEC-HPLC. The method for detecting the VP number of the adenovirus through the SEC-HPLC (size exclusion high performance liquid chromatography) method comprises the step of quantitatively detecting the VP number of the adenovirus through the SEC-HPLC method, wherein a chromatographic column adopted by the SEC-HPLC method is a TSK-gel gel chromatographic column, and a mobile phase is a Tris-HCl NaCl solution; the method can be used for quantitatively detecting the number of the adenovirus VP in virus harvesting liquid and stock solution. The method for detecting the VP number of the adenovirus through the SEC-HPLC method has the advantages of being easy to operate, good in repeatability and high in precision.

Description

Technical Field

[0001] The present invention relates to the field of virus detection, and particularly to a method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC. Background Art

[0002] Adenovirus (Adv) is a double-stranded DNA non-enveloped virus with virus particles having a diameter of 70-90 nm, which are formed by arranging 252 capsomeres in an icosahedral arrangement.

[0003] Traditional virus vaccines are mainly inactivated vaccines or attenuated vaccines, and their safety and effectiveness can be further improved and developed. With the development of genetic engineering, using modified virus vectors to express and induce antigen genes with immunoprotective effects has become an important approach and means for vaccine development.

[0004] The advantages of adenovirus vectors include a wide host range and the ability to infect dividing and non-dividing cells. The virus genome is relatively stable and it is easy to produce high-titer viruses. It is convenient to insert larger foreign DNA fragments, and at the same time, mutations of inserted genes can be reduced. The process is relatively simple, facilitating rapid manufacturing and having a relatively low economic cost. It is relatively safe for humans and has relatively high immunogenicity.

[0005] Initially, adenovirus vectors were mainly applied to gene therapy, especially tumor therapy. Due to the strong innate and adaptive immune responses induced by adenovirus self-antigens, the effect of human gene transfer through adenovirus vectors, including replacing missing or defective genes, is not good. However, this advantage of adenovirus vectors themselves makes them candidate vectors for expressing foreign genes and inducing strong innate and adaptive immune responses. This provides an opportunity for the development of genetic engineering vaccines based on adenovirus vectors and has been widely used in the research of live vector vaccines.

[0006] Most adenoviruses only cause mild diseases in the population. By deleting the early promoter transcription region on the adenovirus genome, replication-deficient attenuated adenovirus vectors can be produced and prepared, thereby reducing the potential side effects caused by the body's infection with live viruses. Adenovirus vector vaccines can not only be administered intramuscularly, but also can be used for mucosal immunization by oral administration, nasal spray and other methods. With the continuous development of adenovirus vectors in the vaccine field, it is crucial to develop a method for stably, rapidly and accurately quantifying the number of adenovirus VPs. Currently, fluorescence quantitative PCR (QPCR) is used to amplify adenovirus conserved genes (such as hexon genes) to achieve quantitative detection of adenoviruses. However, the pretreatment steps of the QPCR method are complex, with many operations, high prices, long cycles, poor repeatability, and the gene homology between different adenovirus serotypes is low, making it difficult to design broad-spectrum primers and requiring the use of multiplex PCR or low-stringency conditions, which may reduce specificity. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a method for quantitatively detecting the VP number of adenovirus based on SEC-HPLC. The method is an adenovirus VP detection method with simple operation, high sensitivity and good repeatability.

[0008] The technical solution of the present invention is as follows: A method for quantitatively detecting the VP number of adenovirus based on SEC-HPLC, the method comprising the following steps: The adenovirus test sample is pretreated with nuclease and then separated and determined by SEC-HPLC, the chromatogram is recorded, and a linear equation is obtained, and the VP number of the adenovirus in the test sample solution is calculated using the linear equation;

[0009] The chromatographic column used in the method is a gel chromatographic column;

[0010] The mobile phase used in the method is:

[0011] Phase A: Tris-HCl solution;

[0012] Phase B: Tris-HCl NaCl solution.

[0013] As a further description of the above solution: The detailed operation of pretreating the adenovirus test sample with nuclease is as follows: The adenovirus harvest fluid sample is treated with nuclease, the nuclease concentration is 10-100 U / mL, the treatment time is between 0-4 hours, and after completion, the nuclease is terminated with a solution of 15-25 mM Tris and 5-15 mM EDTA at pH 6.8-8.0, and then filtered through a 0.22 μm filter membrane and loaded onto the column.

[0014] Preferably, the number of virus particles in the adenovirus test sample is not less than 3.5x10 8 VP / mL. If the number of virus particles is too low, it is difficult to detect and quantify by this method.

[0015] As a further description of the above solution: The concentration of the Tris-HCl solution in Phase A is 0.01-0.05 mol / L; the concentration of Tris-HCl in Phase B of Tris-HCl NaCl is 0.01-0.05 mol / L, and the concentration of NaCl is 0.5-1.5 mol / L. The mobile phase not only maintains the structural stability of adenovirus, but also inhibits non-specific adsorption, improving the peak shape symmetry and detection repeatability.

[0016] As a further description of the above solution: The mixture of mobile phases A and B is used as the mobile phase, the pH range of the mobile phase is 7.0-8.5, the flow rate is 0.3-1.0 mL / min, the column temperature is 20-35 °C, and the detection wavelengths are 260 nm and 280 nm.

[0017] As a further description of the above solution: The specifications of the gel chromatography column are as follows: the column length ranges from 50 to 300 mm, the inner diameter of the chromatography column ranges from 1 to 10 mm, and the particle size ranges from 10 to 30 μm.

[0018] As a further description of the above solution: The column length of the gel chromatography column is 300 mm, the inner diameter of the chromatography column is 7.8 mm, and the particle size is 13 μm.

[0019] As a further description of the above solution: The chromatography column is a gel chromatography column, specifically: TSK-gel G6000PWxl; its pore size highly matches the adenovirus particles (diameter 70 - 90 nm), and can achieve efficient separation of the virus and impurities. The mobile phase is: a mixed solution of 20 mM Tris-HCl in phase A, 20 mM Tris-HCl and 1.0 M NaCl in phase B, and its ratio is: phase A: 55%, phase B: 45%.

[0020] As a further description of the above solution: The adenovirus sample is adenovirus harvest fluid, stock solution, and adenovirus intermediates from R & D or production purification. Among them, the stock solution and some intermediates have been treated with nuclease during the purification process and do not require re-nuclease treatment before detection; the concentration range of the adenovirus harvest fluid is between 5E+9 - 1.5E+10 VP / mL.

[0021] The principle of detecting adenovirus in the present invention is as follows: SEC-HPLC (size exclusion high performance liquid chromatography) is an HPLC technique based on separation by molecular size. It uses stationary phases (fillers) with different pore sizes to separate proteins in the sample, enabling large molecules to pass through the chromatography column before small molecules, thereby achieving separation and purity analysis of proteins. The molecular weight of adenovirus is 1.75*10 8 Da, and its molecular weight is much larger than other substances in the sample system (such as: host proteins). Therefore, this application selects the SEC-HPLC method for the separation and quantification of adenovirus. This method has the characteristics of simple operation, high sensitivity, and good repeatability, and can achieve rapid quantification of adenovirus.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The SEC-HPLC method provided by the present invention directly quantifies the number of VPs by physically separating intact virus particles, avoiding defects such as complex pretreatment operations, long detection cycles, and low throughput. It is particularly suitable for rapid quality control of the virus production process (such as stock solution, harvest fluid), filling the gap in the demand for high-precision and high-throughput detection in industrial production.

[0023] A method for quantitatively detecting the VP number of adenovirus by SEC-HPLC provided by the present invention can effectively separate adenovirus and impurities due to the detection of virus by SEC-HPLC, achieving the detection purpose. Moreover, the operation is simple, with small human error and high accuracy; it can realize real-time monitoring of the VP number of virus stock solution / harvest solution and rapid evaluation of batch-to-batch consistency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the chromatogram of the adenovirus harvest solution without nuclease treatment;

[0025] Figure 2 It is the chromatogram of the adenovirus harvest solution after nuclease treatment;

[0026] Figure 3 It is the chromatogram of the adenovirus harvest solution treated with 10 - 100 U / mL nuclease;

[0027] Figure 4 It is the chromatogram of the adenovirus harvest solution treated for 1 - 4 hours;

[0028] Figure 5 It is the adenovirus standard curve;

[0029] Figure 6 It is the chromatogram of the adenovirus limit of quantification;

[0030] Figure 7 It is the chromatogram of the adenovirus harvest solution;

[0031] Figure 8 It is the chromatogram of the adenovirus stock solution sample. SPECIFIC EMBODIMENTS

[0032] To deepen the understanding of the present invention, the following will further elaborate on the present invention in combination with specific embodiments and drawings. This embodiment is only used to explain the present invention, but the present invention is not limited to the following technical solutions.

[0033] In the following embodiments, Tris-HCl refers to a buffer solution of tris(hydroxymethyl)aminomethane and hydrochloric acid, Tris-HClNaCl refers to a mixed solution of Tris-HCl and sodium chloride, and SEC-HPLC refers to size exclusion high performance liquid chromatography. E+7, E+8, E+9, E+10, and E+11 appearing in the present invention respectively represent 10 7 , 10 8 , 10 9 , 10 10 , and 10 11 .

[0034] According to the detection method described in the present invention, mainly for the detection of the VP number of adenovirus, a linear equation is used for calculation.

[0035] The instrument and reagent information used in the implementation of the detection method of the present invention is shown in Table 1 and Table 2.

[0036] Table 1 Instrument Information

[0037] Name Model Manufacturer Electronic balance 17252 Mettler pH meter Model S470 Mettler Centrifuge Micro21R Thermo Fisher High performance liquid chromatograph ACCHROMS6000 Huapu Scientific Instrument Co., Ltd.

[0038] Table 2 Reagent Information

[0039]

[0040]

[0041] Example 1 Specificity

[0042] 1. Detection method

[0043] High performance liquid chromatograph: Huapu Keyi ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0044] (1) Preparation of mobile phase

[0045] Prepare 0.02 mol / L Tris-HCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02 mol / L Tris-HCl and 1.0 mol / L NaCl solution, adjust the pH to 8.0 as mobile phase B.

[0046] (2) Preparation of test solution

[0047] Take adenovirus samples 1, 2, and 3, divide each sample into 2 parts. One part is centrifuged (8000 rpm, 2 - 8 °C, 2 min), filtered through a 0.22 μm filter membrane to obtain the test solution. The other part of the sample is treated with nuclease, reacted at room temperature for 1 hour, then centrifuged (8000 rpm, 2 - 8 °C, 2 min), and filtered through a 0.22 μm filter membrane to obtain the test solution.

[0048] 2. Chromatographic conditions

[0049] Chromatographic column model: TSK-gel G6000PWxl (7.8 * 300 mm).

[0050] Mobile phase: Mobile phase A - Mobile phase B (55:45)

[0051] Flow rate: 0.5 mL / min

[0052] Column temperature: 25 °C

[0053] Detection wavelength: 260 nm

[0054] Table 3 Specificity

[0055]

[0056]

[0057] From Figure 1 and Figure 2 the results in Table 3, it can be seen that after adding nuclease to the adenovirus harvest fluid sample, its resolution can be effectively improved.

[0058] 3. Screening of nuclease treatment conditions

[0059] 3.1 Nuclease concentration

[0060] Take the adenovirus harvest fluid sample (concentration: 9.8E+10 VP / mL), divide it into 5 equal parts on average, add nuclease to make its final concentration 0 U / mL, 10 U / mL, 30 U / mL, 60 U / mL, 100 U / mL, react at room temperature for 1 hour, then use a solution with a final concentration of 20 mM Tris 10 mM EDTA pH 6.8 - 8.0 to terminate the nuclease reaction, and filter through a 0.22 um filter membrane to be ready for sample injection and detection. The results are shown in Figure 3 .

[0061] From Figure 3 the results, it can be seen that when the nuclease concentration is 10 - 100 U / mL, the resolution between adenovirus and the adjacent peak can reach 1.2. From the integral value, the difference is within 5%, and this difference is within the acceptable range. Currently, from the experimental results, nuclease concentrations of 10 - 100 U / mL can all effectively treat nucleic acids.

[0062] 3.2 Nuclease treatment time

[0063] Take the adenovirus harvest fluid sample, divide it into 5 equal parts on average, add nuclease to make its final concentration 100 U / mL, treat it at room temperature for 0, 1, 2, 3, 4 hours, then add a solution with a final concentration of 20 mM Tris 10 mM, EDTA pH 6.8 - 8.0 to terminate the nuclease digestion reaction, and filter through a 0.22 um filter membrane to be ready for sample injection and detection. The results are shown in Figure 4 .

[0064] From Figure 4 the results, it can be seen that after the nuclease treatment time reaches more than 1 hour, the resolution of adenovirus can reach 1.2. There are differences in the integral values for different treatment times, and the error is within 5%, which belongs to the acceptable range. Therefore, considering comprehensively, the optimal nuclease treatment time is 1 hour.

[0065] Example 2

[0066] 1. Detection method

[0067] High-performance liquid chromatograph: Huapu Keyi ACCHROM S6000 high-performance liquid chromatography system and chemical workstation.

[0068] (1) Mobile phase preparation

[0069] Prepare 0.02 mol / L Tris-HCl solution, and adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02 mol / L Tris-HCl and 1.0 mol / L NaCl solution, and adjust the pH to 8.0 as mobile phase B.

[0070] (2) Test solution preparation

[0071] Take the adenovirus stock solution sample, centrifuge (8000 rpm, 2 - 8 °C, 2 min), and filter through a 0.22 μm filter membrane to obtain the test solution.

[0072] (3) Reference solution preparation

[0073] Take the adenovirus reference product, perform serial dilution, and filter through a 0.22 μm filter membrane.

[0074] 2. Chromatographic conditions

[0075] Chromatographic column model: TSK-gel G6000PWxl (7.8 * 300 mm);

[0076] Mobile phase: Mobile phase A - Mobile phase B (55:45);

[0077] Flow rate: 0.5 mL / min;

[0078] Column temperature: 25 °C;

[0079] Detection wavelength: 260 nm.

[0080] Respectively take the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and obtain the linear equation. Use the linear equation to calculate the number of adenovirus VPs in the test solution. The results are shown in Table 4 and Figure 5 . From Figure 5 It can be seen that the accuracy and reliability of the obtained standard curve are relatively good.

[0081] Table 4 Adenovirus linear data table

[0082] Adenovirus reference product 3.63E+11 3.63E+11 3.63E+11 3.63E+11 3.63E+11 Sampling volume (ul) 5 15 30 60 100 Loading volume (mL) 0.1 0.1 0.1 0.1 0.1 Concentration after dilution (VP / mL) 7.26E+09 2.18E+10 4.36E+10 8.71E+10 1.45E+11 Peak area at 260 nm 292336 914094 1973958 4217263 7174468 Loading amount (VP) 7.26E+08 2.18E+09 4.36E+09 8.71E+09 1.45E+10

[0083] Example 3

[0084] 1. Detection method

[0085] High performance liquid chromatograph: Huapu Keyi ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0086] (1) Mobile phase preparation

[0087] Prepare a 0.02 mol / L Tris-HCl solution and adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare a 0.02 mol / L Tris-HCl and 1.0 mol / L NaCl solution and adjust the pH to 8.0 as mobile phase B.

[0088] (2) Preparation of test solution

[0089] Take the adenovirus harvest fluid sample (concentration: 7.5E+10 VP / mL), add nuclease at a final concentration of 100 U / mL and incubate for 1 hour. After incubation, terminate the nuclease with a solution of 20 mM Tris and 10 mM EDTA at pH 6.8 - 8.0, centrifuge (8000 rpm, 2 - 8 °C, 2 min), and filter through a 0.22 μm filter membrane to obtain the test solution.

[0090] (3) Preparation of reference solution

[0091] Take the adenovirus reference product, perform serial dilution, and filter through a 0.22 μm filter membrane.

[0092] Separatel y take the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and obtain the linear equation. Use the linear equation to calculate the number of adenovirus VPs in the test solution.

[0093] 2. Chromatographic conditions

[0094] Chromatographic column model: TSK-gel G6000PWxl (7.8 * 300 mm).

[0095] Mobile phase: Mobile phase A - Mobile phase B (55:45);

[0096] Flow rate: 0.6 mL / min;

[0097] Column temperature: 25 °C;

[0098] Detection wavelength: 260 nm.

[0099] Example 4

[0100] 1. Detection method

[0101] High performance liquid chromatograph: Huapu Keyi ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0102] (1) Preparation of mobile phase

[0103] Prepare a 0.02 mol / L Tris-HCl solution and adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare a 0.02 mol / L Tris-HCl and 1.0 mol / L NaCl solution and adjust the pH to 8.0 as mobile phase B.

[0104] (4) Preparation of test sample solution

[0105] (5) Take the adenovirus harvest solution sample (concentration: 7.5E+10 VP / mL), add nuclease at a final concentration of 100 U / mL and incubate for 1 hour. After incubation, terminate the nuclease digestion reaction with a solution of 20 mM Tris and 10 mM EDTA at pH 6.8 - 8.0. Centrifuge (8000 rpm, 2 - 8 °C, 2 min), and filter through a 0.22 μm filter membrane to obtain the test sample solution.

[0106] (6) Preparation of reference sample solution

[0107] Take the adenovirus reference sample, perform serial dilution, and filter through a 0.22 μm filter membrane.

[0108] Separate the reference sample solution and the test sample solution, inject them into the liquid chromatograph, record the chromatogram, and obtain the linear equation. Use the linear equation to calculate the number of adenovirus VPs in the test sample solution.

[0109] 2. Chromatographic conditions

[0110] Chromatographic column model: TSK-gel G6000PWxl (7.8 * 300 mm).

[0111] Mobile phase: Mobile phase A - Mobile phase B (50:50)

[0112] Flow rate: 0.6 mL / min

[0113] Column temperature: 25 °C

[0114] Detection wavelength: 260 nm

[0115] Example 5

[0116] 1. Detection method

[0117] High performance liquid chromatograph: Huapu Keyi ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0118] (1) Preparation of mobile phase

[0119] Prepare 0.02 mol / L Tris-HCl solution, and adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02 mol / L Tris-HCl and 1.0 mol / L NaCl solution, and adjust the pH to 8.0 as mobile phase B.

[0120] (7) Preparation of test sample solution

[0121] Take the adenovirus harvest fluid sample (concentration: 7.5E+10 VP / mL), add nuclease at a final concentration of 100 U / mL and process for 1 hour. After completion, terminate the nuclease with a solution of 20 mM Tris 10 mM EDTA pH 6.8 - 8.0, centrifuge (8000 rpm, 2 - 8 °C, 2 min), and filter through a 0.22 um filter membrane to obtain the test solution.

[0122] (8) Preparation of reference solution

[0123] Take the adenovirus reference product, serially dilute it, and filter through a 0.22 um filter membrane.

[0124] Separate the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and obtain the linear equation. Use the linear equation to calculate the number of adenovirus VPs in the test solution.

[0125] 2. Chromatographic conditions

[0126] Chromatographic column model: TSK-gel G6000PWxl (7.8 * 300 mm).

[0127] Mobile phase: Mobile phase A - Mobile phase B (50:50)

[0128] Flow rate: 0.6 mL / min

[0129] Column temperature: 30 °C

[0130] Detection wavelength: 260 nm

[0131] According to the chromatographic conditions provided in Examples 2 - 5, inject the sample to detect adenovirus and obtain the chromatogram.

[0132] The differences of adenovirus under different chromatographic conditions are shown in Table 5.

[0133] Table 5 Differences of adenovirus under different chromatographic conditions

[0134]

[0135] As can be seen from the above table, in the above 4 examples, adenovirus is not interfered by other substances, and the RSD is 0.26%, less than 0.2%, indicating good specificity.

[0136] Example 6 Quantitative limit and detection limit

[0137] The quantitative limit and detection limit are determined according to the signal-to-noise ratio method. The reference solution is serially diluted, the measured signal is compared with the baseline noise, and the lowest concentration that may be detected is calculated. The concentration when the signal-to-noise ratio is about 10 is the quantitative limit concentration, and the concentration when the signal-to-noise ratio is about 3 is the detection limit concentration. The results are shown in Table 6 and Figure 6 .

[0138] Table 6 Results of limit of quantitation and limit of detection

[0139]

[0140] As can be seen from the above table, the limit of detection of adenovirus VP number is 7.02E+07 VP / mL, and the limit of quantitation is 3.51E+08 VP / mL, indicating that the sensitivity of this method is relatively high.

[0141] Example 7 Linearity and range

[0142] Take the adenovirus reference product and prepare solutions diluted 5-fold, 15-fold, 30-fold, 60-fold, and 200-fold respectively as the test solutions for each linear gradient. Plot the measured peak area integral against the injected VP number to obtain the linear regression equation. It is required that the value of the linear regression coefficient R 2 is not less than 0.999. The results are shown in Table 7.

[0143] Table 7 Results of linearity determination

[0144]

[0145] As can be seen from the above table, the linear correlation coefficient R 2 for the detection of adenovirus VP number by the method of the present invention is 0.9997, indicating a good linear relationship.

[0146] Example 8 Precision detection of adenovirus reference product solution

[0147] Take the adenovirus reference product solution and measure it continuously for 6 times to examine the relative standard deviation of the peak area. The results are shown in Table 8.

[0148] Table 8 Results of injection precision experiment of adenovirus reference product solution

[0149]

[0150] As can be seen from the above results, for the injection precision of the adenovirus reference product solution by the detection method of the invention, the RSD of the peak area is less than 2%, indicating good precision.

[0151] Example 9 Stability detection of test solution

[0152] Take the adenovirus harvest fluid and the stock solution samples and inject them at 0 h, 3 h, 12 h, and 24 h respectively. Record the chromatograms and calculate the relative standard deviation of the adenovirus peak area. The results are shown in Table 9.

[0153] Table 9 Results of stability detection of reference product and test solution

[0154] Time Peak area of harvest fluid Peak area of stock solution 0h 1906047 4311944 3h 1877415 4426668 12h 1859998 4319767 24h 1891669 4306001 Average value 1883782 4306001 RSD 1.05％ 1.33％

[0155] As can be seen from the above table, for the adenovirus harvest fluid and bulk sample, when stored at 2-8°C for 24 hours, the RSD of the peak areas of the virus harvest fluid and bulk sample are both less than 2%, indicating good solution stability.

[0156] Repeatability test of Example 10

[0157] Take the adenovirus harvest fluid and bulk sample, and repeat the injection 6 times according to the detection method of Example 3 to verify the good precision of the method. The results are shown in Table 10 and Figure 7 - Figure 8 .

[0158] Table 10 Results of repeatability experiment

[0159] Number Peak area of adenovirus harvest fluid Peak area of adenovirus stock solution 1 7538361 3699298 2 7163544 3631805 3 7266062 3667873 4 7184493 3719419 5 7188623 3762594 6 7263571 3771319 Average value 7267442 3658718 RSD 1.92％ 1.46％

[0160] As can be seen from the above table, the RSD of the adenovirus peak area for 6 injections is less than 2, proving that the method has good precision.

[0161] Accuracy test of Example 11

[0162] The spiking recovery method was used for determination. The ratio between the actual measured value and the theoretical value of adenovirus in the spiked sample was determined and expressed as a percentage. The recovery rate was required to be between 80% and 120% to confirm that the method has good accuracy. The results are shown in Table 11.

[0163] Table 11 Results of accuracy experiment

[0164]

[0165]

[0166] As can be seen from the above table, the spiking recovery rates of adenovirus in the harvest fluid and bulk sample are between 80% and 120%, meeting the verification requirements, confirming that the method has good accuracy, and the RSD of the recovery rate is less than 10, indicating good repeatability.

[0167] In summary, in the specificity experiment, the impurity peaks of the test sample do not affect the detection of adenovirus, and the resolution is good.

[0168] In the quantitative limit and detection limit experiments, the quantitative limit is 3.51E+8 VP / mL, and the detection limit is 7.01E+7 VP / mL, proving that this method has high sensitivity.

[0169] In the linearity and range test, the linear correlation coefficient R of adenovirus 2 is 0.9997, proving that this method shows a good linear relationship.

[0170] In the repeatability experiment, the RSD of the peak areas for 6 sample injections is less than 2.

[0171] In the accuracy experiment, the recovery rate of adenovirus was between 80% and 120%, and the RSD was less than 10.

[0172] It can be seen therefrom that the method of the present invention can effectively isolate adenovirus and perform accurate quantification, and at the same time shows incomparable advantages in terms of specificity, limit of quantification, limit of detection, linearity, accuracy and repeatability, and has high precision.

[0173] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should be regarded as within the protection scope of the present invention.

Claims

1. A method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC, characterized in that The method includes the following steps: the adenovirus test sample is pretreated with nuclease and then separated and determined by SEC-HPLC. Record the chromatogram and obtain the linear equation, and use the linear equation to calculate the number of adenovirus VPs in the test sample solution; The chromatographic column used in the method is a gel chromatographic column; The mobile phase used in the method is: Phase A: Tris-HCl solution; Phase B: Tris-HCl NaCl solution.

2. The method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC according to claim 1, wherein The adenovirus test sample is treated with nuclease. The nuclease concentration is 10-100 U / mL, and the treatment time is 0-4 hours. After the treatment, the nuclease is terminated with a solution with a final concentration of 15-25 mM Tris and 5-15 mM EDTA at pH 6.8-8.0, and then filtered through a filter membrane and loaded onto the column.

3. The method for quantitatively detecting the adenovirus VP number based on SEC-HPLC according to claim 1, wherein The number of virus particles in the adenovirus test sample is not less than 3.5x10 8 VP / mL.

4. The method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC according to claim 1, wherein The concentration of the Tris-HCl solution in Phase A is 0.01-0.05 mol / L; the concentration of Tris-HCl in Tris-HCl NaCl in Phase B is 0.01-0.05 mol / L, and the concentration of NaCl is 0.5-1.5 mol / L.

5. The method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC according to claim 1, wherein The mixture of mobile phases A and B is used as the mobile phase. The pH range of the mobile phase is 7.0-8.5, the flow rate is 0.3-1.0 mL / min, the column temperature is 20-35 °C, and the detection wavelengths are 260 nm and 280 nm.

6. The method for quantitatively detecting adenovirus VP number based on SEC-HPLC according to claim 1, characterized in that, The specifications of the gel chromatographic column are: the column length is between 50-300 mm, the inner diameter of the chromatographic column is between 1-10 mm, and the particle size is between 10-30 μm.

7. The method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC according to claim 1, wherein The column length of the gel chromatographic column is 300 mm, the inner diameter of the chromatographic column is 7.8 mm, and the particle size is 13 μm.

8. The method for quantitatively detecting the number of adenovirus VPs based on SEC-HPLC according to claim 1, wherein The chromatographic column is a gel chromatographic column, specifically: TSK-gel G6000PWxl; the mobile phase is: Phase A 20 mM Tris-HCl, and Phase B is a mixed solution of 20 mM Tris-HCl and 1.0 M NaCl, and the ratio is: Phase A: 55%, Phase B: 45%.

9. The method for quantitatively detecting the adenovirus VP number based on SEC-HPLC according to claim 1, wherein The adenovirus test sample is an adenovirus harvest solution, a stock solution, and an adenovirus intermediate derived from R & D or production purification.

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