Method for detecting purity of recombinant adenovirus preparation

Abstract

The invention relates to the technical field of biological detection, in particular to a purity detection method of a recombinant adenovirus preparation. According to the method, high performance liquid chromatography is adopted for detection, an ion exchange chromatographic column filled with Proteomix POR-Q is used for separating the purity of the recombinant adenovirus with non-replicating Ad5 as a carrier with negative charges on the surface in a preparation, and the peak area of a target object is quantitatively analyzed through an ultraviolet detector, so that the detection result has good specificity and accuracy; according to the method, the detected mobile phase, elution procedure, column temperature and other parameters are obtained through optimization, so that the quantitative result has good precision, a new reliable method is provided for quality control of the recombinant adenovirus preparation, and standardized popularization, use and quality monitoring of the recombinant adenovirus preparation are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting the purity of a recombinant adenovirus preparation. Background Art

[0002] Viral purity in recombinant adenovirus preparations refers to the purity of the target viral particles in the preparation, that is, the ratio of target viral particles to impurities (such as incompletely packaged viral particles, incorrectly packaged viral particles, inactive viral particles, viral particle aggregates, free viral genomes, host cell proteins, etc.). As an important tool for gene therapy and vaccine delivery, recombinant adenovirus purity testing is a key step in ensuring safety and efficacy.

[0003] High-purity adenovirus preparations can retain their natural antigenic epitope structure by maintaining the complete spatial conformation of the viral capsid protein. This characteristic has important clinical significance. First, the reduction in the impurity content in the preparation can effectively reduce nonspecific immune activation, enabling the body's immune system to more accurately identify and respond to target antigens. Secondly, high-purity preparations significantly reduce the proportion of non-infectious viral particles, avoiding the competitive inhibition of these defective particles on the effective infection process, thereby ensuring a more accurate correspondence between therapeutic doses and clinical effects. More importantly, this high-purity characteristic ensures a high degree of consistency between different production batches, providing a solid foundation for the reproducibility of clinical efficacy, which is particularly important for the standardized application of gene therapy products.

[0004] Existing methods for assessing viral purity include ultracentrifugation, flow cytometry, transmission electron microscopy, ultraviolet spectrophotometry, and SDS-PAGE. While these methods can analyze adenovirus purity to varying degrees, they are subject to numerous interference factors, poor accuracy, and inability to accurately quantify the amount of adenovirus.

[0005] High-performance liquid chromatography (HPLC) combines the separation mechanism of chromatographic columns with highly sensitive detectors to accurately quantify viral purity. SEC-HPLC and IEX-HPLC are the mainstream methods, and the choice should be adjusted based on the characteristics of the virus (size, charge) and the type of impurities. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting the purity of a recombinant adenovirus preparation.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The present invention provides a method for detecting the purity of a recombinant adenovirus preparation, which adopts high performance liquid chromatography for detection. The chromatographic column of the high performance liquid chromatography is an ion exchange chromatographic column filled with Proteomix POR-Q.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, the specification of the ion exchange chromatography column is 15 μm, 4.6×50mm.

[0011] Furthermore, in the high-performance liquid chromatography method, the injection volume is 20-30 μl, the detection column temperature is 25-35° C., and the temperature of the sample to be tested is 3-8° C.

[0012] Furthermore, the mobile phase A of the high performance liquid chromatography method is a Tris solution, and the mobile phase B is a mixed solution of Tris and sodium chloride.

[0013] Furthermore, in the mobile phase A, the concentration of Tris is 15-25 mmol / l, and the pH value is 7-8; in the mobile phase B, the concentration of Tris is 15-25 mmol / l, the concentration of sodium chloride is 0.5-1.5 mmol / l, and the pH value is 7-8.

[0014] Furthermore, the injection volume was 25 μl, the column temperature was 30°C, and the temperature of the sample to be tested was 5°C; in the mobile phase A, the concentration of Tris was 20 mmol / l and the pH value was 7.5; in the mobile phase B, the concentration of Tris was 20 mmol / l, the concentration of sodium chloride was 1 mmol / l, and the pH value was 7.5.

[0015] Furthermore, the high-performance liquid chromatography method adopts a gradient elution method for detection. Taking the total volume of the mobile phase A and the mobile phase B as 100%, the elution procedure is:

[0016] Time (min) Flow rate (mL / min) Mobile phase A% Mobile phase B% 0 0.5 100 0 10 0.5 100 0 10.001 0.5 84 16 14 0.5 84 16 17 0.5 84 16 17.001 0.5 65 35 19 0.5 65 35 22 0.5 65 35 22.001 0.5 50 50 24 0.5 50 50 26 0.5 50 50 26.001 0.5 0 100 27 0.5 0 100 33 0.5 0 100 33.001 0.5 100 0 40 0.5 100 0

[0017] Furthermore, the detection wavelength of the high performance liquid chromatography is 260 nm and 280 nm, and the retention time is 24.5-24.9 min.

[0018] Further, the following steps are included:

[0019] Performing the high performance liquid chromatography test on the sample to be tested and the internal control product of known purity respectively;

[0020] Confirm the system suitability of the method based on the test results of the internal control product;

[0021] The purity of the virus in the sample to be tested is obtained according to the peak area detection result of the sample to be tested.

[0022] Furthermore, the recombinant adenovirus in the sample to be tested is a non-replicating Ad5 vector with a negative surface charge.

[0023] The beneficial effects of the present invention are:

[0024] (1) The purity detection method of the recombinant adenovirus preparation of the present invention utilizes an ion exchange chromatography column filled with Proteomix POR-Q to detect the purity of the recombinant adenovirus containing a negatively charged surface non-replicating Ad5 as a carrier in the preparation, so that the qualitative detection results have good specificity and accuracy;

[0025] (2) The purity detection method of the recombinant adenovirus preparation of the present invention optimizes the detection parameters such as the mobile phase, elution program, and column temperature, so that the quantitative results have good precision;

[0026] (3) The purity detection method of the recombinant adenovirus preparation of the present invention provides a new and reliable method for the quality control of the recombinant adenovirus preparation, which is helpful for the standardized promotion, use and quality monitoring of the recombinant adenovirus preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the chromatogram of the first experimental group in Example 1, a method for detecting the purity of a recombinant adenovirus preparation of the present invention;

[0028] Figure 2 This is the chromatogram of the second experimental group in Example 1, a method for detecting the purity of a recombinant adenovirus preparation of the present invention;

[0029] Figure 3 This is the chromatogram of the third experimental group in Example 1, which is a method for detecting the purity of the recombinant adenovirus preparation of the present invention;

[0030] Figure 4 This is the chromatogram of the fourth experimental group in Example 1, which is a method for detecting the purity of the recombinant adenovirus preparation of the present invention;

[0031] Figure 5 This is the purity detection method of the recombinant adenovirus preparation of the present invention, and the chromatogram of the Source15Q chromatographic column in Example 2;

[0032] Figure 6 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of the Saifen chromatographic column in Example 2;

[0033] Figure 7 This is the purity detection method of the recombinant adenovirus preparation of the present invention, and the chromatogram of the Agilent chromatographic column in Example 2;

[0034] Figure 8 This is a chromatogram of the first injection sample of the first experimental group in Example 3, showing the purity detection method of the recombinant adenovirus preparation of the present invention;

[0035] Figure 9 This is the purity detection method of the recombinant adenovirus preparation of the present invention, and the chromatogram of the first injection sample of the second experimental group in Example 3;

[0036] Figure 10 This is the purity detection method of the recombinant adenovirus preparation of the present invention, and the chromatogram of the blank control group in Example 4;

[0037] Figure 11 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of the sample group in Example 4;

[0038] Figure 12 This is the chromatogram of the impurity interference group in Example 4 of the purity detection method of the recombinant adenovirus preparation of the present invention;

[0039] Figure 13 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of RE-1 in Example 5;

[0040] Figure 14 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of LOQ 9-1 in Example 6;

[0041] Figure 15 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of LOQ 6-1 in Example 6;

[0042] Figure 16 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of LOQ 2-1 in Example 6;

[0043] Figure 17 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of T24-1 in Example 7;

[0044] Figure 18 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of IP-4 in Example 5;

[0045] Figure 19 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of T28-1 in Example 7;

[0046] Figure 20 The purity detection method of the recombinant adenovirus preparation of the present invention is shown in the chromatogram of T32-1 in Example 7;

[0047] Figure 21This is the chromatogram of 20230705AH01 obtained by using the purity detection method described in the literature "Preliminary Study on Quality Control, Large-Scale Amplification and Purification of Adenovirus CNHK200-hEndostatin by Xue Huibin" in Example 1;

[0048] Figure 22 This is the chromatogram of 20230705AH01 obtained by using the purity detection method in the document "Preliminary Study on Quality Control, Large-Scale Amplification and Purification of Adenovirus CNHK200-hEndostatin by Xue Huibin" in Example 1. DETAILED DESCRIPTION

[0049] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0050] The purity detection method of the recombinant adenovirus preparation of the present invention adopts high performance liquid chromatography for detection, and the chromatographic column of the high performance liquid chromatography is an ion exchange chromatography column filled with Proteomix POR-Q.

[0051] The purity detection method of the present invention utilizes an ion exchange chromatography column filled with Proteomix POR-Q to detect the purity of a recombinant adenovirus containing a negatively charged surface non-replicating Ad5 vector in a preparation. By optimizing the detection parameters such as the mobile phase, elution program, and column temperature, the detection results have good specificity, accuracy, and precision, providing a new and reliable method for quality control of recombinant adenovirus preparations.

[0052] Ion exchange chromatography is a commonly used purification method in protein purification technology. Its principle is that the charge carried by the substance to be separated can bind to the opposite charge carried by the ion exchanger. This binding interaction between the charged molecule and the stationary phase is reversible. When the pH is changed or eluted with a buffer with gradually increasing ionic strength, the substance bound to the ion exchanger can be exchanged with the ions in the eluent and eluted into the solution. Due to the different charges of different substances, their binding ability with the ion exchanger is also different, so they are eluted into the solution in a different order, and thus separated. The hexameric protein, the main component of the adenovirus shell, is negatively charged under neutral conditions. Therefore, the surface of the virus particle carries a large amount of negative charge, making anion exchange the first choice for detecting adenovirus purity.

[0053] Preferably, the specification of the ion exchange chromatography column of the present invention is 15 μm, 4.6×50mm.

[0054] Preferably, in the high-performance liquid chromatography method of the present invention, the injection volume is 20-30 μl, the detection column temperature is 25-35° C., and the temperature of the sample to be tested is 3-8° C.

[0055] Preferably, the mobile phase A of the high performance liquid chromatography method of the present invention is a Tris solution, and the mobile phase B is a mixed solution of Tris and sodium chloride.

[0056] Further preferably, in mobile phase A, the concentration of Tris is 15-25 mmol / l, and the pH value is 7-8; in mobile phase B, the concentration of Tris is 15-25 mmol / l, the concentration of sodium chloride is 0.5-1.5 mmol / l, and the pH value is 7-8.

[0057] Further preferably, the injection volume is 25 μl, the column temperature for detection is 30°C, and the temperature of the sample to be tested is 5°C; in the mobile phase A, the concentration of Tris is 20 mmol / l, and the pH value is 7.5; in the mobile phase B, the concentration of Tris is 20 mmol / l, the concentration of sodium chloride is 1 mmol / l, and the pH value is 7.5.

[0058] Preferably, the high performance liquid chromatography method adopts a gradient elution method for detection, and the total volume of mobile phase A and mobile phase B is 100%, and the elution procedure is:

[0059] Time (min) Flow rate (mL / min) Mobile phase A% Mobile phase B% 0 0.5 100 0 10 0.5 100 0 10.001 0.5 84 16 14 0.5 84 16 17 0.5 84 16 17.001 0.5 65 35 19 0.5 65 35 22 0.5 65 35 22.001 0.5 50 50 24 0.5 50 50 26 0.5 50 50 26.001 0.5 0 100 27 0.5 0 100 33 0.5 0 100 33.001 0.5 100 0 40 0.5 100 0

[0060] Preferably, the detection wavelength of the high performance liquid chromatography is 260 nm and 280 nm, and the retention time is 24.5-24.9 min.

[0061] The detection method of the present invention specifically comprises the following steps:

[0062] The sample to be tested and the internal control product of known purity were tested by high performance liquid chromatography respectively;

[0063] Confirm the system suitability of the method based on the test results of internal control products;

[0064] The purity of the virus in the sample to be tested is calculated based on the peak area detection result of the sample to be tested.

[0065] The present invention is illustrated below by means of specific examples.

[0066] Example 1 Screening of HPLC Elution Program

[0067] This embodiment first adopts existing chromatography detection, and the specific conditions are as follows:

[0068] Mobile phase A: 20 mM Tris pH 8.0 solution;

[0069] Mobile phase B: 20 ​​mM Tris 0.1 M NaCl pH 8.0 solution;

[0070] Chromatographic column: Resource Q (4.6 × 100 mm) column;

[0071] Flow rate: 1 ml / min, detection wavelength: 260 nm.

[0072] Elution gradient:

[0073] Time (min) Mobile phase A Mobile phase B 0 100 0 21 30 70 30 100 0

[0074] The chromatogram of this method is as follows Figure 21 and Figure 22 shown. Figure 21 Specifically, the purity test method described in the literature "Preliminary Study on Quality Control, Large-Scale Amplification and Purification of Adenovirus CNHK200-hEndostatin by Xue Huibin" was used to test the chromatogram of sample 20230705AH01; Figure 22 This is the chromatogram of sample 20230705AH01 detected using the purity detection method in the document "Preliminary Study on Quality Control, Large-Scale Amplification and Purification of Adenovirus CNHK200-hEndostatin by Xue Huibin".

[0075] Figure 21 and Figure 22 In the figure, the blue curve represents sample 20230705AH01, and the black curve represents the blank solvent. It was found that the sample eluted too early, not binding to the column. Since high-purity KDTV001 samples are currently unavailable, both KDTV001 and KD01 samples are adenovirus particles, and the principles used in this method are the same. We will consider using high-purity KD01 samples for main peak localization in the future.

[0076] The titer is 1×10 10TG6 at the 100 ng / ml level was used as a high-purity sample of KD01. The results of high-performance liquid chromatography (HPLC) of four elution procedures were compared to screen for the optimal elution procedure. TG6 is from Wuhan Kaidejinuo Biotechnology Co., Ltd. For more information about KD01, please refer to patent number CN 117568405A, "A recombinant oncolytic adenovirus vector, construction method, and application thereof." The E1A region of the KD01 gene is missing from the 920th to 946th nt region, and the E3 region is missing from the 29483rd to 29721st nt region of the ADP gene. The gene sequence for the mitochondrial apoptotic peptide tBid was inserted into the missing region, and a ClaI restriction site was introduced. The KD01 genome is approximately 30-38 kb in size, and is a viral particle with a diameter of 80-110 nm. The capsid is an icosahedron composed of 240 hexons and 12 pentons. After testing, the retention time of TG6 is about 29 minutes. According to the characteristics of biological products and referring to the relevant parameters of the HPCL method for purity detection of biological products in the Chinese Pharmacopoeia, the system suitability standards of this method are set as follows: the number of theoretical plates should be ≥5000, the resolution should be ≥1.5, and the tailing factor / asymmetry should be ≤2.

[0077] Since the chromatographic column used in the prior art is a Source 15Q chromatographic column, this embodiment also uses this chromatographic column for experiments.

[0078] The elution conditions for each experimental group in this example were as follows: mobile phase A: 20 mM Tris pH 7.5 solution, mobile phase B: 20 ​​mM Tris 1 M NaCl pH 7.5 solution, flow rate: 0.5 ml / min, detection wavelength: 260 nm.

[0079] The elution procedures for each experimental group were as follows Figure 1-4 and as shown in Table 1-4.

[0080] Table 1 Elution procedure of the first experimental group

[0081]

[0082]

[0083] Table 2 Elution procedure of the second experimental group

[0084] T(min) Mobile phase A% Mobile phase B% curve 0 100 0 N / A 10 100 0 6 14 84 16 1 17 84 16 6 19 65 35 1 22 65 35 6 24 50 50 1 26 50 50 6 27 35 65 1 30 35 65 6 31 28 72 1 33 28 72 6 33.1 100 0 1 40 100 0 6

[0085] Table 3 Elution procedure of the third experimental group

[0086]

[0087]

[0088] Table 4 Elution procedure of the fourth experimental group

[0089] T(min) Mobile phase A% Mobile phase B% curve 0 100 0 N / A 10 100 0 6 14 84 16 1 17 84 16 6 19 65 35 1 22 65 35 6 24 50 50 1 26 50 50 6 50 0 100 6 50.1 100 0 1 55 100 0 6

[0090] The system applicability of the above four experimental groups was compared, and the comparison results are shown in Table 5.

[0091] Table 5 Comparison of system applicability of each experimental group

[0092] method Theoretical plates Separation tailing factor The first experimental group 173711 9.49 1.40 The second experimental group 176413 9.47 1.40 The third experimental group 9627 6.42 1.31 The fourth experimental group 6285 5.76 1.35

[0093] As can be seen from Table 5, under the elution conditions of the first experimental group, the number of theoretical plates was 173711, the resolution was 9.49, and the tailing factor was 1.40. Under the elution conditions of the second experimental group, the number of theoretical plates was 176413, the resolution was 9.47, and the tailing factor was 1.40. The system applicability of the two methods was not much different. Under the elution conditions of the third experimental group, the number of theoretical plates was 9627, the resolution was 6.42, and the tailing factor was 1.31. Under the elution conditions of the fourth experimental group, the number of theoretical plates was 6285, the resolution was 5.76, and the tailing factor was 1.35. The system applicability of the latter two methods was poor.

[0094] Therefore, the elution conditions of the first experimental group were the best.

[0095] Example 2 Screening and Verification of Different Chromatographic Columns

[0096] The self-assembled Source 15Q chromatographic column used in the prior art and Example 1 exhibits poor stability. To ensure the stability of subsequent testing, this example investigated different chromatographic columns to screen for suitable commercially available columns and ensure the stability of the method. This example also used KD01 sample batch number TG6.

[0097] In this example, three chromatographic columns, Source 15Q (self-assembled), Proteomix POR-Q, and Agilent Bio-Monolith Column QA, were initially selected for comparative testing. The system suitability comparison of the three chromatographic columns is shown in Table 6. The chromatograms are shown in Table 6. Figure 5-7 shown.

[0098] Table 6 Comparison of applicability of different chromatographic column systems

[0099] Chromatographic columns Theoretical plates Separation tailing factor Source15Q 173711 9.49 1.40 Saifen chromatography column 62702 1.85 1.97 Agilent columns 13075 4.76 2.66

[0100] As shown in Table 6, the tailing factor in the Agilent column system suitability does not meet the requirements, so the Agilent column was excluded. Further confirmation was conducted on the Source 15Q and Separ columns, which met the system suitability requirements.

[0101] The experiment was repeated three times for Source 15Q and Selfin columns, and the column data comparison is shown in Table 7.

[0102] Table 7 Column data comparison

[0103]

[0104] It can be seen from Table 7 that the results of both the Saifen column and Source 15Q meet the experimental requirements. Since the Source15Q column is a self-assembled column, considering the differences in filling by personnel, the Saifen column is the preferred column for this method.

[0105] In addition, considering that different chromatographs also have an impact on the test results, this embodiment also uses Waters (USP) and Thermo (EP) equipment to perform tests. The test results are shown in Table 8.

[0106] Table 8 System suitability and sample purity results of different equipment

[0107]

[0108] It can be seen that the system suitability of both devices meets the requirements, and the system suitability and results of the two experiments are in line with the experimental requirements, indicating that the elution procedure and chromatographic column of this method have good stability.

[0109] Example 3 Qualitative Effect Verification of the Method

[0110] This example verifies the qualitative effect of the method of the present invention, specifically using different samples of known purity for detection.

[0111] Specifically, the samples used in this embodiment are all KDTV001. KDTV001 is an HPV therapeutic vaccine. The virus contained therein is a non-replicating type 5 adenovirus containing HPV16 / 18 / 52 type E6 / E7 antigens. The E1 and E3 regions of the non-replicating type 5 adenovirus are missing. For the specific sequence and information of KDTV001, please refer to patent CN202410543158 "A HPV16_18_52 therapeutic vaccine, preparation method and application".

[0112] The sample to be tested in the first experimental group of this embodiment is KDTV00120240328AH03, and the sample to be tested in the second experimental group is KDTV00110L scale stock solution (LOT: DS240822). The purity of both is 100%. Each experimental group is injected three times in parallel. The system suitability and sample purity test values ​​of the two experimental groups are shown in Tables 9 and 10. The chromatogram of the first injection sample of the first experimental group is shown in Table 9. Figure 8 As shown, the chromatogram of the first injection sample of the second experimental group is as follows Figure 9shown.

[0113] Table 9 The first experimental group

[0114]

[0115]

[0116] Table 10 Second experimental group

[0117]

[0118] After testing, the sample peaks and impurity peaks in the two experimental groups can be completely separated, the system applicability meets the requirements, the number of theoretical plates is ≥5000, the separation degree is ≥1.5, and the asymmetry is ≤2, indicating that the method of the present invention can be used to detect the purity of the virus in the KDTV001 product.

[0119] Example 4 Method Specificity Verification

[0120] In this example, the KDTV001 adenovirus storage solution (V) was used as a blank control group, the KDTV001 stock solution was used as a sample group, and a mixed solution of the KDTV001 stock solution and HEK293 HCP was used as an impurity interference group. The methods of the present invention were used to test each of these solutions to verify the specificity of the present invention. The volume ratio of KDTV001 to HEK293 HCP in the impurity interference group was 9:1.

[0121] KDTV001 adenovirus storage solution (V) is a sample with no interfering peaks at the target peak position of the KDTV001 product. The purity of the KDTV001 stock solution is 100%, the resolution is N / A, and the mixed solution of KDTV001 stock solution and HEK293HCP has a purity of 95.14% and a resolution of 11.38.

[0122] In this embodiment, each of the above groups was tested in parallel once, and the obtained results were processed using a 3D quantitative method to check the sample purity and the separation between the main peak and the impurity peak. The results are shown in Table 11 and the chromatogram is shown in Figure 10-12 shown.

[0123] Table 11

[0124]

[0125] It has been verified that the KDTV001 adenovirus preservation solution (V) of the blank control group had no interfering peak at the target peak position of the KDTV001 product, the main peak purity of the KDTV001 stock solution sample group was 100%, the main peak purity of the impurity interference group was 95.14%, and the separation degree R between the main peak and the protein impurity peak was 11.38, which met the requirements (no interfering peak at the stock solution peak position; main peak purity ≥95%; separation degree R between the main peak and impurity peak ≥1.5).

[0126] Example 5 Method Precision Verification

[0127] This example performs precision verification on the method of the present invention, specifically including verification of repeatability and intermediate precision.

[0128] (1) Repeatability: The same experimenter took the same batch of KDTV001 stock solution 6 times in a row and measured the purity of its target peak. Each time, one injection of sample was made. The repeatability of the method was investigated by calculating the RSD of the test results. The results are shown in Table 12. The chromatogram of RE-1 is shown in Figure 13 shown.

[0129] Table 12

[0130]

[0131] According to the above results, it can be seen that the RSD of the target peak purity of the test sample in the 6 experimental results of the repeatability test is 0%, and the RSD of the main peak area is 3.62%, which meets the requirements (RSD of the target peak purity of the test sample ≤ 2%; RSD of the main peak area ≤ 6.0%).

[0132] (2) Intermediate precision: The same batch of KDTV001 stock solution was used by different analysts, using different batches of mobile phase, different batches of chromatographic columns, and three separate measurements at different times. One sample was injected each time to determine the purity of the target peak. The RSD of the 12 test results was calculated. The results are shown in Table 13. The chromatogram of IP-4 is shown in Table 13. Figure 18 shown.

[0133] Table 13

[0134]

[0135] According to the results, it can be seen that the RSD of the target peak purity of the test sample in the 12 test results of the intermediate precision test is 0%, which meets the requirements (RSD of the target peak purity of the test sample ≤ 2%).

[0136] Example 6 Determination of Quantitation Limit, Detection Limit and Detection Range

[0137] This example investigates the quantitative effect of the method by determining the quantitative limit, detection limit, and detection range of the method.

[0138] Specifically, in this example, 1 mL of KDTV001 stock solution was added to 9 mL of adenovirus storage solution for a total of 10-fold dilution, which served as the limit of quantification (LOQ) -1. Subsequently, the dilution was continued in a 2-fold gradient until LOQ -9 was reached. The number of viral particles in each experimental group is shown in Table 14. The limit of quantification for the purity assay was determined as the percentage of the main peak area relative to the 100% main peak area when the main peak signal-to-noise ratio was ≥10 and the precision met the requirements. The limit of detection for the purity assay was determined as the percentage of the main peak area relative to the 100% main peak area when the main peak signal-to-noise ratio was ≥3 and the precision met the requirements. The test results are shown in Table 15.

[0139] The chromatograms of LOQ 9-1, LOQ 6-1 and LOQ 2-1 are as follows Figure 14-16 As shown. It has been verified that the SN values ​​of all samples tested at LOQ-1 to LOQ-9 were ≥10, but the RSD of the peak area of ​​three parallel injections of samples at the three concentrations of LOQ-7, LOQ-8, and LOQ-9 was >6.0%. This does not meet the repeatability requirements. Therefore, the limit of quantification and the limit of detection of the purity test method are both LOQ-6, that is, the minimum peak area is 0.273 and the number of viral particles is 2.81×10 9 VP / ml.

[0140] The lowest range of the quantitative limit of purity is the percentage of the main peak area relative to the main peak area of ​​100% concentration. Therefore, it can be determined that the purity range of the sample to be tested by the detection method of the present invention is 0.63% to 100%; the number of virus particles is 2.81×10 9 -9×10 11 VP / ml. This detection range can basically cover the situations in actual applications.

[0141] Table 14

[0142] Sample name Particle count (VP / ml) Original solution <![CDATA[9.00×10 11 ]]> LOQ-1 <![CDATA[9.00×10 10 ]]> LOQ-2 <![CDATA[4.50×10 10 ]]> LOQ-3 <![CDATA[2.25×10 10 ]]> LOQ-4 <![CDATA[1.13×10 10 ]]> LOQ-5 <![CDATA[5.63×10 9 <!-- 11 -->]]> LOQ-6 <![CDATA[2.81×10 9 ]]> LOQ-7 <![CDATA[1.41×10 9 ]]> LOQ-8 <![CDATA[7.03×10 8 ]]> LOQ-9 <![CDATA[3.52×10 8 ]]>

[0143] Table 15

[0144]

[0145]

[0146] Example 7 Method Durability Verification

[0147] This example examines the durability of this method from two aspects: sample stability and column temperature conditions.

[0148] (1) Sample stability: The KDTV001 stock solution was tested using the detection method of the present invention. Specifically, the KDTV001 stock solution was placed at 2-8°C and sampled immediately at 0 h, 24 h, and 48 h for target peak purity. Three parallel experiments were performed, with one injection per parallel experiment. The test results are shown in Table 16. The specific requirement for sample stability is that the recovery rate of the detection values ​​at the 24 h and 48 h storage time points relative to the initial 0 h detection value (main peak area and purity) must be between 80% and 120%.

[0149] Table 16

[0150]

[0151]

[0152] The chromatogram of T0-1 is shown in Figure 13 The chromatogram of T24-1 is as follows: Figure 17 As shown, the chromatogram of T48-1 is shown in Figure 18 According to the test results, the recovery rate of the main peak area of ​​the detection value at the 24-hour placement time point relative to the initial 0-hour detection value is 109.78%, and the purity recovery rate is 100%; the recovery rate of the main peak area of ​​the detection value at the 48-hour placement time point relative to the initial 0-hour detection value is 82.78%, and the purity recovery rate is 100%, which meets the requirements.

[0153] (2) Column temperature conditions: The detection method of the present invention was used to detect the KDTV001 stock solution. Specifically, the KDTV001 stock solution was taken, and the column temperature was set to 28°C, 30°C, and 32°C, respectively. The target peak purity was detected by loading the sample separately. Three samples were prepared in parallel under each column temperature condition, and one injection was injected into each sample. The recovery rates of the two temperature points relative to the detection values ​​(main peak area and purity) at the column temperature of 28°C and 32°C should be between 80% and 120% relative to the detection values ​​at the 30°C point. The test results are shown in Table 17.

[0154] Table 17

[0155]

[0156] The chromatogram of T30-1 can be found in Figure 13 The chromatogram of T28-1 is as follows: Figure 19 As shown, the chromatogram of T32-1 is as follows Figure 20 As shown in the test results, the recovery rate of the main peak area at a column temperature of 28°C relative to the detection value at 30°C is 113.41%, and the purity recovery rate is 100%. The recovery rate of the main peak area at a column temperature of 32°C relative to the detection value at 30°C is 115.49%, and the purity recovery rate is 100%, which meets the requirements.

[0157] It can be seen that good detection results are obtained when the samples are injected at different times and detected at different column temperatures, indicating that the method of the present invention has good stability.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the purity of a recombinant adenovirus preparation, characterized in that: The detection was performed by high performance liquid chromatography, wherein the chromatographic column of the high performance liquid chromatography was an ion exchange chromatography column filled with Proteomix POR-Q.

2. The method for detecting the purity of a recombinant adenovirus preparation according to claim 1, wherein: The specification of the ion exchange chromatography column is 15 μm. 4.6×50mm.

3. The method for detecting the purity of a recombinant adenovirus preparation according to claim 2, wherein: In the high-performance liquid chromatography method, the injection volume is 20-30 μl, the detection column temperature is 25-35° C., and the temperature of the sample to be tested is 3-8° C.

4. The method for detecting the purity of a recombinant adenovirus preparation according to claim 3, wherein: The mobile phase A of the high performance liquid chromatography method is a Tris solution, and the mobile phase B is a mixed solution of Tris and sodium chloride.

5. The method for detecting the purity of a recombinant adenovirus preparation according to claim 4, wherein: In the mobile phase A, the concentration of Tris is 15-25 mmol / l, and the pH value is 7-8. In the mobile phase B, the concentration of Tris is 15-25 mmol / l, the concentration of sodium chloride is 0.5-1.5 mmol / l, and the pH value is 7-8.

6. The method for detecting the purity of a recombinant adenovirus preparation according to claim 5, wherein: The injection volume was 25 μl, the column temperature was 30°C, and the temperature of the sample to be tested was 5°C. In the mobile phase A, the concentration of Tris was 20 mmol / l and the pH value was 7.

5. In the mobile phase B, the concentration of Tris was 20 mmol / l, the concentration of sodium chloride was 1 mmol / l, and the pH value was 7.

5.

7. The method for detecting the purity of a recombinant adenovirus preparation according to claim 6, wherein: The high-performance liquid chromatography method adopts a gradient elution method for detection. Taking the total volume of the mobile phase A and the mobile phase B as 100%, the elution procedure is as follows:

8. The method for detecting the purity of a recombinant adenovirus preparation according to claim 7, wherein: The detection wavelength of the high performance liquid chromatography is 260 nm and 280 nm, and the retention time is 24.5-24.9 min.

9. The method for detecting the purity of a recombinant adenovirus preparation according to any one of claims 1 to 8, wherein: The following steps are involved: Performing the high performance liquid chromatography test on the sample to be tested and the internal control product of known purity respectively; Confirm the system suitability of the method based on the test results of the internal control product; The purity of the virus in the sample to be tested is calculated based on the peak area detection result of the sample to be tested.

10. The method for detecting the purity of a recombinant adenovirus preparation according to claim 9, wherein: The recombinant adenovirus in the sample to be tested is a non-replicating Ad5 vector with a negative charge on the surface.

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