A primer probe composition for detecting genetic stability of cells and application thereof

Abstract

The application provides a primer probe composition for detecting genetic stability of cells and application thereof, the primer probe composition comprises primer pairs SEQ ID NO:1-2 and a probe SEQ ID NO:3 of a VEGF light chain gene, and further comprises primer pairs SEQ ID NO:4-5 and a probe SEQ ID NO:6 of a VEGF heavy chain gene. The application quantitatively detects the antibody light and heavy chain gene copy numbers in cells of different culture generations by fluorescence quantitative PCR based on a Taqman probe method, extracts cell genomic DNA and performs PCR amplification, and prepares a target gene standard product from the amplification product, thereby significantly improving the specificity and accuracy of quantitative detection of the gene copy number.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and gene detection technology. It relates to a primer and probe composition for detecting the genetic stability of cells and its application, and more particularly to a primer and probe composition, kit, detection method and its application for detecting the genetic stability of recombinant CHO cells. Background Technology

[0002] TaqMan probe assay is a widely used quantitative real-time PCR (qPCR) method. Compared to conventional PCR, the TaqMan probe-based PCR reaction system includes not only primers, dNTPs, DNA polymerase, and template, but also probes with fluorescent and quenching groups at both ends, approximately 20–25 nt in length. This method utilizes the 5'–3' exonuclease properties of DNA polymerase. During PCR extension after annealing, the DNA polymerase gradually cleaves the probe, creating a spatial distance between the 5' fluorescent group and the 3' quenching group. At a specific excitation wavelength, the fluorescence emitted by the fluorescent group is not absorbed by the quenching group, resulting in fluorescence emission. By collecting the fluorescence intensity during the PCR process, a linear relationship between nucleic acid content and cycle number is fitted to standards and test samples, thereby determining the content of the target gene in the test sample.

[0003] CHO cells, or Chinese hamster ovary cells, are widely used in the preparation of monoclonal antibodies and recombinant protein biopharmaceuticals. These cells possess advantages such as a clear genetic background, a well-developed expression system, and high protein expression levels. When expressing exogenous proteins using CHO cells, the target gene needs to be introduced into the nucleus of the CHO cell using a suitable expression vector, integrated into the CHO cell's chromosome, and then processed through large-scale culture, purification, and formulation to produce suitable drugs or reagents. During the passage of transgenic cells, the genetic stability of the target gene is a crucial factor. The 2015 edition of the Chinese Pharmacopoeia, Volume III, General Rules for Biological Products, "Preparation and Testing Procedures for Animal Cell Substrates for the Production and Testing of Biological Products," explicitly states the specific requirements for recombinant cells: "Producers must have data on the stability of the target gene used in production, including the genetic stability of the recombinant cells (such as the copy number of the inserted gene)."

[0004] Currently, existing literature reports methods for detecting the copy number of recombinant monoclonal antibodies in CHO cells, using both the digital droplet PCR method (CN105368957A) and the SYBR Green method (CN106191216A). The advantages of digital droplet PCR are that it eliminates the need for standard preparation and offers high detection sensitivity. However, its disadvantages include the high cost of the equipment and reagents (a single digital droplet PCR instrument can cost up to 3 million RMB), and a relatively narrow linear range, typically between 2 and 20,000 copies / PCR reaction. The SYBR Green method, based on the binding characteristics of fluorescent dyes to double-stranded DNA, is simple and low-cost. However, non-specific amplification of DNA products and primer dimers can interfere with the quantitative results, leading to lower accuracy.

[0005] Compared to the two methods above, the Taqman probe method has the advantages of lower cost, higher specificity of template amplification, and a wider linear range of standard curve, generally between 10 and 10,000,000 copies / PCR reaction.

[0006] CN102154503A discloses a probe-based method for detecting CHO cell DNA content. Specific primer pairs and TaqMan probes are designed using the nucleotide sequence of GeneBank accession number EF540878.1. After extracting DNA from the sample, real-time quantitative PCR is performed. Based on the obtained standard curve, the amount of CHO cell DNA in the sample is calculated, achieving the goal of accurately quantifying the residual CHO cell DNA content in therapeutic protein drugs, recombinant vaccines, and monoclonal antibodies derived from CHO cells. However, this method is intended for detecting residual CHO cell DNA content, and an internal reference gene is added during the quantification of the target gene, achieving relative quantification. The quantification result of the internal reference gene can significantly affect the quantification result of the target gene.

[0007] Therefore, it is of great significance to provide a Taqman probe-based PCR quantification method with higher accuracy, stronger specificity, wider linear range, and lower cost to identify the genetic stability of cell lines during large-scale cell culture. Summary of the Invention

[0008] To address the shortcomings of existing technologies and practical needs, this invention provides a primer-probe composition for detecting cellular genetic stability and its application. The kit and detection method constructed therefrom utilize the TaqMan probe method to quantitatively detect the copy number of the light and heavy chains of recombinant monoclonal antibodies during passage in CHO cells, examine the genetic stability of transgenic exogenous proteins, and provide data support for the stability of cell lines in large-scale cell culture.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a primer-probe composition for detecting cellular genetic stability, the primer-probe composition comprising primer pairs SEQ ID NO:1-2 and probe SEQ ID NO:3 of the VEGF (vascular endothelial growth factor) light chain gene;

[0011] SEQ ID NO:1: upstream primer for VEGF light chain gene

[0012] 5'-GCTGCACCATCTGTCTTCATCT-3';

[0013] Downstream primer for the VEGF light chain gene, SEQ ID NO:2:

[0014] 5'-GCACACAACAGAAGCAGTTCCA-3';

[0015] VEGF light chain gene probe SEQ ID NO:3:

[0016] 5'-CCCGCCATCTGATGAGCAGTTGAA-3'.

[0017] Preferably, the primer-probe composition further includes primer pairs SEQ ID NO:4-5 and probe SEQ ID NO:6 of the VEGF heavy chain gene;

[0018] The upstream primer for the VEGF heavy chain gene is SEQ ID NO:4:

[0019] 5'-CAGCCGGAGAACAACTACAAGA-3';

[0020] Downstream primer for the VEGF heavy chain gene SEQ ID NO:5:

[0021] 5'-GAGCTTGCTGTAGAGGAAGAAGGA-3';

[0022] VEGF heavy chain gene probe SEQ ID NO:6:

[0023] 5'-CACGCCTCCCGTGCTGGACTC-3'.

[0024] In this invention, upstream and downstream primers and probes designed based on the VEGF light chain gene and heavy chain gene are beneficial for quantitatively detecting the copy number of recombinant monoclonal antibody light and heavy chains in CHO cells during passage, and for identifying cell genetic stability.

[0025] In a second aspect, the present invention provides a kit for detecting cellular genetic stability, the kit comprising the primer-probe composition as described in the first aspect.

[0026] Preferably, the kit further includes target gene standards, which include VEGF light chain gene standards and / or VEGF heavy chain gene standards.

[0027] In this invention, the preparation process of the target gene standard involves amplifying cellular genomic DNA using VEGF light chain gene primer pairs or VEGF heavy chain gene primer pairs, then inserting the amplified VEGF light chain gene or VEGF heavy chain gene into a plasmid via TA cloning to construct plasmid DNA. After plasmid extraction, the concentration and purity of the plasmid are detected using a spectrophotometer, and the copy number (copies / μL) of the standard is calculated using a formula, which serves as the target gene standard.

[0028] This invention extracts genomic DNA from cells and performs PCR amplification, then prepares target gene standards from the amplification products, thereby improving the specificity and accuracy of quantitative detection of gene copy numbers.

[0029] Preferably, the concentration of the VEGF light chain gene standard is 1×10⁻⁶. 2 ~1×10 7 Copy number / μL, for example, could be 1×10 2 Copy number / μL, 1×10 3 Copy number / μL, 1×10 4 Copy number / μL, 1×10 5 Copy number / μL, 1×10 6 Copy number / μL or 1×10 7 Copy number / μL.

[0030] Preferably, the concentration of the VEGF heavy chain gene standard is 1×10⁻⁶. 2 ~1×10 7 Copy number / μL, for example, could be 1×10 2 Copy number / μL, 1×10 3 Copy number / μL, 1×10 4 Copy number / μL, 1×10 5 Copy number / μL, 1×10 6 Copy number / μL or 1×10 7 Copy number / μL.

[0031] Preferably, the kit further includes DNA polymerase, PCR buffer, and dNTPs.

[0032] Preferably, the kit further includes a negative control comprising ultrapure water.

[0033] Thirdly, the present invention provides a method for developing a standard curve for a reagent kit as described in the second aspect, comprising the following steps:

[0034] The target gene standard was serially diluted, and quantitative real-time PCR was performed using the primer and probe composition described in the first aspect to obtain the CT value.

[0035] A standard curve was constructed with CT values ​​on the ordinate and the logarithm of the copy number of the target gene standard on the abscissa.

[0036] Preferably, the reaction conditions for the real-time PCR are 48–52℃ for 1–3 min; 92–98℃ for 8–12 min; 92–98℃ for 10–20 s; 58–62℃ for 0.8–1.2 min, for a total of 38–42 cycles.

[0037] Preferably, the formula for calculating the copy number of the target gene standard is: target gene standard concentration × Avogadro's constant / target gene standard molecular weight.

[0038] Fourthly, the present invention provides a method for detecting cellular genetic stability, comprising the following steps:

[0039] CT values ​​were obtained by performing real-time PCR on cellular genomic DNA using the primer and probe composition as described in the first aspect or the kit as described in the second aspect.

[0040] The copy number of cellular genomic DNA is calculated based on the standard curve and CT value.

[0041] In this invention, quantitative real-time PCR based on the Taqman probe method is used to quantitatively detect the copy number of antibody light and heavy chain genes in cells at different culture passages, which significantly improves the detection specificity and accuracy and helps to monitor the quality of recombinant protein drug production process.

[0042] Preferably, the cellular genomic DNA is derived from cells at different culture passages.

[0043] Preferably, the cells comprise CHO cells.

[0044] Preferably, the fluorescence quantitative PCR reaction conditions are 48-52℃ for 1-3 min; 92-98℃ for 8-12 min; 92-98℃ for 10-20 s; 58-62℃ for 0.8-1.2 min, for a total of 38-42 cycles.

[0045] Fifthly, the present invention provides the use of the primer-probe composition as described in the first aspect and / or the kit as described in the second aspect in the preparation of protein drugs.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) This invention uses quantitative real-time PCR based on Taqman probe method to quantitatively detect the copy number of antibody light and heavy chain genes in cells of different culture generations, extracts genomic DNA from cells and performs PCR amplification, and prepares target gene standards from the amplification products, which significantly improves the specificity and accuracy of gene copy number quantitative detection, with a CV% of less than 12% between replicates.

[0048] (2) The method for detecting cell genetic stability of the present invention has high accuracy, strong specificity, wide linear range and low cost, which helps to monitor the quality of recombinant protein drug production process and has broad application prospects in the industrial production of recombinant proteins. Attached Figure Description

[0049] Figure 1 This is a qPCR amplification pattern of the light chain standard curve;

[0050] Figure 2 For light chain standard curves;

[0051] Figure 3 The copy number of the light chain in the genome of CHO cells at different generations;

[0052] Figure 4 This is the qPCR amplification pattern of the heavy chain standard curve;

[0053] Figure 5 This is the standard curve for heavy chains;

[0054] Figure 6 The heavy chain copy number of the genome in CHO cells of different generations. Detailed Implementation

[0055] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0056] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0057] Example 1: Preparation of VEGF light chain gene standard

[0058] (1) Preparation of light chain plasmid DNA

[0059] Primer pairs as shown in SEQ ID NO:1-2 and probes as shown in SEQ ID NO:3 were designed based on the VEGF light chain gene.

[0060] Genomic DNA was extracted from CHO cells using Invitrogen's Purelink Genomic DNA kit. The complete VEGF light chain gene was amplified using primer pairs VEGFLF (SEQ ID NO:1) and VEGFLR (SEQ ID NO:2). The PCR reaction system was as follows: 2×PrimeSTAR Max Premix, 25 μL; VEGFLF (10 nM), 1 μL; VEGFLR (10 nM), 1 μL; ddH2O, 21 μL; genomic DNA, 2 μL. The reaction conditions were: 98℃ for 10 min; 98℃ for 1 min; 57℃ for 5 s; 72℃ for 90 s, for a total of 30 cycles.

[0061] The amplification products were subjected to a poly A-tailing reaction using Taq enzyme. The reaction system was as follows: Taq enzyme, 0.2 μL; 10×Taq enzyme buffer, 1 μL; dNTP (10 mM), 0.5 μL; PCR product, 8.3 μL; reaction conditions: 72℃, 10–15 min.

[0062] The PCR product with a poly A tail was ligated to the pEASY-T3 plasmid via TA, transformed into competent cells, and clones were picked and screened to obtain the pEASY-T3 recombinant plasmid strain containing the light chain gene.

[0063] Recombinant plasmids were extracted using Invitrogen's Purelink Genomic DNA kits. Plasmids that were identified as positive by gel electrophoresis were sent for sequencing. The size of the recombinant plasmids was 3741 bp.

[0064] (2) Preparation of light chain plasmid DNA standards

[0065] The correctly sequenced positive clone strain was inoculated into 2 mL of Amp+ LB liquid medium and cultured in a shaker at 37°C for about 8 hours; when the strain activity was activated and the concentration was high, it was inoculated into 100 mL of Amp+ LB liquid medium at a ratio of 1:100 and cultured in a shaker at 37°C overnight.

[0066] Plasmid DNA was extracted using the endotoxin-free plasmid extraction kit from Tiangen Biotech (Beijing) Co., Ltd., following the instructions. The plasmid was then eluted with 1.5 mL of elution buffer, and the concentration and purity of the obtained plasmid were determined using a BioPhotometer plus nucleic acid and protein analyzer.

[0067] The copy number of the light chain plasmid DNA standard was calculated using the formula based on the measured plasmid DNA concentration.

[0068] Plasmid DNA copy number = plasmid DNA concentration (g / μL) × Avogadro's constant (copies / mol) / plasmid DNA molecular weight (g / mol).

[0069] Example 2: Establishment of a standard curve for the amplification of the VEGF light chain gene

[0070] According to the plasmid copy number formula in step (2) of Example 1, the copy number of 1 μL of light chain plasmid standard was calculated to be 1.95 × 10⁻⁶. 11 Based on the amplification efficiency and CT value, the initial concentration of the standard curve was finally determined to be 1×10⁻⁶. 7 ~1×10 2 Copies / μL, 10-fold serial dilution; prepare corresponding light chain standard DNA templates at different concentrations;

[0071] The total volume of the light chain standard reaction was 20 μL, including 10 μL of Taqman Universal Master Mix II; 0.25 μL of VEGFLF (10 μM); 0.25 μL of VEGFLR (10 μM); 0.5 μL of VEGFLP probe (SEQ ID NO:3); 8 μL of sterile ultrapure water; and 1 μL of light chain standard DNA template.

[0072] The reaction conditions were 50℃ for 2 min; 95℃ for 10 min; 95℃ for 15 s; 60℃ for 1 min, for a total of 40 cycles.

[0073] qPCR amplification pattern as follows Figure 1 As shown, the concentrations from left to right are 1×10⁻⁶. 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL;

[0074] A standard curve was constructed with CT values ​​on the ordinate and the logarithm of the plasmid standard copy number on the x-axis, as shown below. Figure 2 As shown, the amplification efficiency Eff% = 100.593, R 2 =0.998, and the regression equation of the standard curve is: Y = -3.308log[DNA] + 42.252.

[0075] Example 3 Quantitative detection of light chain genes

[0076] Extraction of cellular genomic DNA: CHO cells transfected with the VEGF antibody gene, cultured to passages 1, 5, 10, 15, 20, 25, and 30, were processed at approximately 3 × 10⁻⁶ cells / year. 6 Collect each cell / piece, centrifuge to remove the supernatant, and store the precipitate at -80℃ for later use;

[0077] According to the Purelink Genomic DNA kits instructions, genomic DNA was extracted from the cells of the above passages, and the concentration was determined using a BioPhotometer plus nucleic acid protein analyzer. The genomic DNA was diluted to 1 μg / mL with sterile ultrapure water as the test sample for qPCR, and sterile ultrapure water was used as the NTC control.

[0078] The reaction system and conditions were the same as in Example 2. The standard samples and test samples from Examples 2 and 3 were reacted simultaneously using the same instrument (Applied Biosystems ABI 7500 quantitative PCR instrument). Specifically, the reaction system and conditions for the test samples were as follows:

[0079] The total reaction volume of the test sample (sample) was 20 μL, including 10 μL of Taqman Universal Master Mix II; 0.25 μL of VEGFLF (10 μM); 0.25 μL of VEGFLR (10 μM); 0.5 μL of VEGFLP probe (SEQ ID NO:3); 8 μL of sterile ultrapure water; and 1 μL of DNA template for the test sample.

[0080] The reaction conditions were 50℃ for 2 min; 95℃ for 10 min; 95℃ for 15 s; 60℃ for 1 min, for a total of 40 cycles.

[0081] Under the above PCR conditions, a standard curve was established using a light chain DNA standard template. Based on the linear relationship between CT value and light chain gene copy number, the Applied Biosystems 7500SDS software automatically calculated the VEGF light chain gene copy number in the test sample.

[0082] See results Figure 3 According to Table 1, the amplification efficiency Eff% = 92.564, R 2 =0.997, the regression equation of the standard curve is: Y = -3.514log[DNA] + 40.037, and the RSD% value of light chain gene copy number among 7 different generations was calculated to be 9.0%.

[0083] Table 1. Copy number of light chain genes in the genome of CHO cells at different generations.

[0084] Cell generation VEGF light chain gene copy number (copies / μL) CV% between multiple holes Generation 1 <![CDATA[1.97×10 5 ]]> 4.1％ 5th generation <![CDATA[1.74×10 5 ]]> 5.7％ 10th generation <![CDATA[1.83×10 5 ]]> 8.0％ 15th generation <![CDATA[2.0×10 5 ]]> 0.6％ 20th generation <![CDATA[1.99×10 5 ]]> 5.8％ 25th generation <![CDATA[1.75×10 5 ]]> 11.2％ 30th generation <![CDATA[1.62×10 5 ]]> 6.2％

[0085] Example 4: Preparation of VEGF heavy chain gene standard

[0086] (1) Preparation of heavy chain plasmid DNA

[0087] Primer pairs as shown in SEQ ID NO:4-5 and probes as shown in SEQ ID NO:6 were designed based on the VEGF heavy chain gene.

[0088] Genomic DNA was extracted from CHO cells using Invitrogen's Purelink Genomic DNA kit. The complete VEGF heavy chain gene was amplified using primer pairs VEGFHF (SEQ ID NO:4) and VEGFHR (SEQ ID NO:5). The PCR reaction system was as follows: 2×PrimeSTAR Max Premix, 25 μL; VEGFHF (10 nM), 1 μL; VEGFHR (10 nM), 1 μL; ddH2O, 21 μL; genomic DNA, 2 μL. The reaction conditions were: 98℃ for 10 min; 98℃ for 1 min; 57℃ for 5 s; 72℃ for 90 s, for a total of 30 cycles.

[0089] The amplification products were subjected to a poly A-tailing reaction using Taq enzyme. The reaction system was as follows: Taq enzyme, 0.2 μL; 10×Taq enzyme buffer, 1 μL; dNTP (10 mM), 0.5 μL; PCR product, 8.3 μL; reaction conditions: 72℃, 10–15 min.

[0090] The PCR product with poly A tail was ligated to the pEASY-T3 plasmid via TA, transformed into competent cells, clones were picked and screened, and the pEASY-T3 recombinant plasmid containing the heavy chain gene was obtained.

[0091] Recombinant plasmids were extracted using Invitrogen's Purelink Genomic DNA kits. Plasmids that were identified as positive by gel electrophoresis were sent for sequencing. The size of the recombinant plasmids was 4458 bp.

[0092] (2) Preparation of heavy chain plasmid DNA standards

[0093] The correctly sequenced positive clone strain was inoculated into 2 mL of Amp+ LB liquid medium and cultured in a shaker at 37°C for about 8 hours; when the strain activity was activated and the concentration was high, it was inoculated into 100 mL of Amp+ LB liquid medium at a ratio of 1:100 and cultured in a shaker at 37°C overnight.

[0094] Plasmid DNA was extracted using the endotoxin-free plasmid extraction kit from Tiangen Biotech (Beijing) Co., Ltd., following the instructions. The plasmid was then eluted with 1.5 mL of elution buffer, and the concentration and purity of the obtained plasmid were determined using a BioPhotometer plus nucleic acid and protein analyzer.

[0095] The copy number of the heavy chain plasmid DNA standard was calculated using the formula based on the measured plasmid DNA concentration.

[0096] Plasmid DNA copy number = plasmid DNA concentration (g / μL) × Avogadro's constant (copies / mol) / plasmid DNA molecular weight (g / mol).

[0097] Example 5: Establishment of a standard curve for the amplification of the VEGF heavy chain gene

[0098] According to the plasmid copy number formula in step (2) of Example 4, the copy number of 1 μL of heavy chain plasmid standard was calculated to be 1.42 × 10⁻⁶. 11 Based on the amplification efficiency and CT value, the initial concentration of the standard curve was finally determined to be 1×10⁻⁶. 7 ~1×10 2 Copies / μL, 10-fold serial dilution; prepare corresponding heavy chain standard DNA templates of different concentrations;

[0099] The total volume of the heavy chain standard reaction was 20 μL, including 10 μL of Taqman Universal Master Mix II; 0.25 μL of VEGFHF (10 μM); 0.25 μL of VEGFHR (10 μM); 0.5 μL of VEGFHP probe (SEQ ID NO:6); 8 μL of sterile ultrapure water; and 1 μL of heavy chain standard DNA template.

[0100] The reaction conditions were 50℃ for 2 min; 95℃ for 10 min; 95℃ for 15 s; 60℃ for 1 min, for a total of 40 cycles.

[0101] qPCR amplification pattern as follows Figure 4 As shown, the concentrations from left to right are 1×10⁻⁶. 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL;

[0102] A standard curve was constructed with CT values ​​on the ordinate and the logarithm of the plasmid standard copy number on the x-axis, as shown below. Figure 5 As shown, the amplification efficiency Eff% = 100.387, R 2 =0.998, and the regression equation of the standard curve is: Y = -3.313log[DNA] + 41.481.

[0103] Example 6 Quantitative detection of heavy chain genes

[0104] Extraction of cellular genomic DNA: CHO cells transfected with the VEGF antibody gene, cultured to passages 1, 5, 10, 15, 20, 25, and 30, were processed at approximately 3 × 10⁻⁶ cells / year. 6 Collect each cell / piece, centrifuge to remove the supernatant, and store the precipitate at -80℃ for later use;

[0105] According to the Purelink Genomic DNA kits instructions, genomic DNA was extracted from the cells of the above passages, and the concentration was determined using a BioPhotometer plus nucleic acid protein analyzer. The genomic DNA was diluted to 1 μg / mL with sterile ultrapure water as the test sample for qPCR, and sterile ultrapure water was used as the NTC control.

[0106] The reaction system and reaction conditions were the same as in Example 5. The standard samples and test samples from Examples 5 and 6 were reacted simultaneously using the same instrument (Applied Biosystems ABI 7500 quantitative PCR instrument). Specifically, the reaction system and reaction conditions for the test sample were as follows: the total reaction volume of the test sample (test sample) was 20 μL, including 10 μL of Taqman Universal Master Mix II; 0.25 μL of VEGFHF (10 μM); 0.25 μL of VEGFHR (10 μM); 0.5 μL of VEGFHP probe (SEQ ID NO: 6); 8 μL of sterile ultrapure water; and 1 μL of the test sample DNA template. The reaction conditions were 50℃ for 2 min; 95℃ for 10 min; 95℃ for 15 s; and 60℃ for 1 min, for a total of 40 cycles.

[0107] Under the above PCR conditions, a standard curve was established using heavy chain DNA standard templates. Based on the linear relationship between CT value and heavy chain gene copy number, Applied Biosystems 7500SDS software automatically calculated the VEGF heavy chain gene copy number in the test sample. Results are shown below. Figure 6 According to Table 2, the amplification efficiency Eff% = 95.852, R 2 =0.994, the regression equation of the standard curve is: Y = -3.426log[DNA] + 43.015, and the RSD% value of heavy chain gene copy number among 7 different generations is calculated to be 13.0%.

[0108] Table 2. Copy number of heavy chain gene in CHO cells of different generations.

[0109] Cell generation VEGF light chain gene copy number (copies / μL) CV% between multiple holes Generation 1 <![CDATA[2.84×10 5 ]]> 6.1％ 5th generation <![CDATA[2.37×10 5 ]]> 4.6％ 10th generation <![CDATA[2.48×10 5 ]]> 2.3％ 15th generation <![CDATA[2.92×10 5 ]]> 2.7％ 20th generation <![CDATA[3.05×10 5 ]]> 2.5％ 25th generation <![CDATA[2.38×10 5 ]]> 5.2％ 30th generation <![CDATA[2.25×10 5 ]]> 1.7％

[0110] In summary, this invention employs quantitative real-time PCR based on the Taqman probe method to quantitatively detect the copy number of antibody light and heavy chain genes in cells at different culture passages. Genomic DNA is extracted from the cells and subjected to PCR amplification. The amplification products are then used to prepare target gene standards, which significantly improves the specificity and accuracy of gene copy number quantification. This invention has important application value in the industrial preparation of recombinant proteins.

[0111] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention. SEQUENCE LISTING <110> Dongyao Pharmaceutical Co., Ltd. <120> A primer-probe composition for detecting cellular genetic stability and its application. <130> 20200121 <160> 6 <170> PatentIn version 3.3 <210> 1 <211> twenty two <212> DNA <213> artificial synthesis <400> 1 gctgcaccat ctgtcttcat ct 22 <210> 2 <211> twenty two <212> DNA <213> artificial synthesis <400> 2 gcacacaaca gaagcagttc ca 22 <210> 3 <211> twenty four <212> DNA <213> artificial synthesis <400> 3 cccgccatct gatgagcagt tgaa 24 <210> 4 <211> twenty two <212> DNA <213> artificial synthesis <400> 4 cagccggaga acaactacaa ga 22 <210> 5 <211> twenty four <212> DNA <213> artificial synthesis <400> 5 gagcttgctg tagaggaaga agga 24 <210> 6 <211> twenty one <212> DNA <213> artificial synthesis <400> 6 cacgcctccc gtgctggact c 21

Claims

1. A primer probe composition for detecting genetic stability of cells based on Taqman probe method, characterized by, The primer probe composition comprises a primer pair SEQ ID NO: 1~2 and a probe SEQ ID NO: 3 of the VEGF light chain gene; The primer probe composition further comprises a primer pair SEQ ID NO: 4~5 and a probe SEQ ID NO: 6 of the VEGF heavy chain gene.

2. A kit for detecting genetic stability of cells, characterized by, The kit comprises the primer probe composition of claim 1, and further comprises a target gene standard, which comprises a VEGF light chain gene standard and / or a VEGF heavy chain gene standard.

3. The kit of claim 2, wherein The concentration of the VEGF light chain gene standard is 1 x 10 2 ~1 x 10 7 copies per μL; The concentration of the VEGF heavy chain gene standard is 1 x 10 2 1 x 10 7 copies / μL.

4. The kit of claim 3, wherein The kit further comprises a DNA polymerase, a PCR buffer and dNTP.

5. The kit of claim 4, wherein The kit further comprises a negative control, which comprises ultrapure water.

6. A method for the preparation of a standard curve for a kit according to any one of claims 2-5, characterized in that, The method comprises the following steps: Gradient dilution of the target gene standard, and fluorescence quantitative PCR using the primer probe composition of claim 1 to obtain CT values; Establishing a standard curve with CT values as the ordinate and the logarithm of the copy number of the target gene standard as the abscissa.

7. The method of claim 6, wherein, The reaction condition of the fluorescence quantitative PCR is 48~52℃ 1~3 min; 92~98℃ 8~12 min; 92~98℃ 10~20 s, 58~62℃ 0.8~1.2 min, a total of 38~42 cycles.

8. The method of claim 7, wherein, The calculation formula of the copy number of the target gene standard is target gene standard concentration x Avogadro constant / target gene standard molecular weight.

9. A method for detecting the genetic stability of cells by quantitatively detecting the copy number of light and heavy chain genes of VEGF monoclonal antibodies in different culture passages of cells by fluorescence quantitative PCR based on Taqman probe method, characterized in that, The method comprises the following steps: Fluorescence quantitative PCR of the cell genomic DNA using the primer probe composition of claim 1, or the kit of any one of claims 2-5 to obtain CT values; According to the standard curve and the CT values, the copy number of the cell genomic DNA is calculated.

10. The method of claim 9, wherein, The cell genomic DNA is derived from cells of different culture passages; The cells comprise CHO cells; The reaction condition of the fluorescence quantitative PCR is 48~52℃ 1~3 min; 92~98℃ 8~12 min; 92~98℃ 10~20 s, 58~62℃ 0.8~1.2 min, a total of 38~42 cycles.

11. Use of a primer probe composition according to claim 1 and / or a kit according to any one of claims 2-5, characterized in that, The application is to detect the cell genetic stability of the VEGF monoclonal antibody light and heavy chain gene copy number of cells of different culture passages.

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