Method for detecting host cell DNA (deoxyribonucleic acid) residue of protein polymer

Abstract

The invention belongs to the field of biological medicines, and discloses a protein polymer host cell DNA residue detection method, which comprises: S1, preparing a calibration sample and a blank solution; s2, preparing a negative control sample; s3, preparing a positive control sample; s4, preparing a protein stock solution sample; s5, preparing a standard sample of the protein stock solution; s6, preparing an intracellular protein sample; s7, preparing an intracellular protein labeling sample; s8, the calibration sample is sent to a machine for detection to obtain a standard curve; s9, samples prepared in the steps S2-S7 are sent to a machine to be detected, and CT values are obtained; and S10, evaluating the credibility of the Ct value in the step S9, and if the Ct value is not credible, adjusting parameters and repeating the steps S1-S9. The DNA residue detection method of some examples is high in detection result accuracy.

Description

Technical Field

[0001] The invention belongs to the field of biological medicines, and in particular relates to a method for detecting residual host cell DNA of a protein polymer. Background Art

[0002] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, and urine. They have the characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and easy collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), etc., and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, immune maturation and other processes. They can be used for immune regulation, tissue repair and treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.

[0003] The inventors have discovered that by stimulating MSCs and then lysing them, a protein polymer with nerve repair activity can be separated and purified from intracellular proteins. This protein polymer has achieved significant results in completed animal and clinical trials, especially for ALS, and has a clear potential for drug development, and has now entered the clinical trial stage.

[0004] At the current stage, protein polymers are produced by stimulating MSCs expression under a specific stress state, then lysing MSCs, fully releasing the proteins in the cell tissue to obtain a cell suspension, and then centrifuging the cell suspension at high speed to obtain the supernatant to obtain the protein polymer. Among them, the protein molecules in the protein polymer are easily bound to the DNA molecules in the host cells. The adhesion between the two is strong and the molecular size is similar. It is difficult to completely remove the DNA molecules during the separation process. Therefore, the residual amount of host cell DNA has also become one of the important parameters for characterizing the quality of protein polymer products, and it is also an important indicator for ensuring the safety of drug use. Therefore, the development of a method for detecting the residual amount of host cell DNA in protein polymers is of great significance for analyzing the purity of protein polymers and evaluating the safety of protein polymer use. Summary of the invention

[0005] The object of the present invention is to overcome at least one deficiency of the prior art and provide a method for detecting the residual amount of host cell DNA of protein polymers.

[0006] The technical solution adopted by the present invention is:

[0007] A method for detecting residual host cell DNA of a protein polymer, comprising:

[0008] S1: Add dilution solvent to DNA standard substance to prepare several groups of calibration samples with different DNA molecular concentrations, and send them into fluorescent quantitative PCR instrument for detection to form a standard curve correlating DNA molecular concentration and Ct value;

[0009] S2: adding a sample pretreatment agent into the stock solution product of the protein polymer to prepare a protein stock solution sample;

[0010] S3: adding a sample pretreatment agent and the calibration sample into the protein polymer stock solution product to prepare a protein stock solution spiked sample;

[0011] S4: adding a sample pretreatment agent into the intracellular protein product of the host cell of the protein polymer to prepare an intracellular protein sample;

[0012] S5: adding a sample pretreatment agent and the calibration sample into the intracellular protein product of the host cell of the protein polymer to prepare an intracellular protein spiked sample;

[0013] S6: Design a blank sample, wherein the blank sample is the dilution solvent;

[0014] S7: Design a negative control sample, wherein the negative control sample is the sample pretreatment agent;

[0015] S8: designing a positive control sample, wherein the positive control sample is prepared by mixing the calibration sample with the sample pretreatment agent;

[0016] S9: sending the sample obtained in steps S2-S8 into a fluorescent quantitative PCR instrument for detection to obtain a Ct value; and

[0017] S10: evaluating and judging the credibility of the Ct values ​​measured in step S9, and substituting the credible Ct values ​​into the standard curve one by one to obtain the corresponding DNA molecule concentration of the sample;

[0018] The evaluation and judgment of the Ct value includes: comparing the Ct values ​​of the negative control sample, the blank sample and the calibration sample with the lowest concentration; when the Ct values ​​of the negative control sample and the blank sample are greater than the Ct value of the calibration sample with the lowest concentration, it is determined that the Ct values ​​determined in step S9 are credible; when at least one of the Ct values ​​of the negative control sample and the blank sample is less than the Ct value of the calibration sample, it is determined that the Ct value determined in step S9 is not credible, and the parameters need to be adjusted to repeat the above steps S1-S9.

[0019] Several groups of calibration samples with different DNA molecule concentrations are prepared and sent to a fluorescent quantitative PCR instrument to measure the Ct value and generate a standard curve. The detection objects include two types of products of the protein polymer: intracellular proteins of host cells and protein stock solutions that have been subjected to corresponding purification treatments for the intracellular proteins. For the above two types of products to be tested that may have certain differences in DNA molecule concentrations, intracellular protein samples and intracellular protein spiked samples, as well as protein stock solution samples and protein stock solution spiked samples are prepared respectively. In addition, negative control samples and positive control samples are designed as controls. By simply comparing the Ct values ​​measured by the negative control samples, blank samples and the calibration samples with the lowest DNA molecule concentration, the credibility of the detection values ​​of other samples in the detection method is determined, the accuracy of the DNA residue detection results is improved, and at the same time, direction is provided for the parameter adjustment of the samples to obtain relatively more accurate DNA residue detection results.

[0020] In some embodiments, the fluorescence quantitative PCR instrument is a fluorescence quantitative PCR instrument of model Quant Studio 5 produced by ABI, the diluent is a DNA diluent, and the sample pretreatment agent is a 10× cell / virus lysis kit with item number AN19L050 produced by Heyuan Liji (Shanghai) Biotechnology Co., Ltd. Professional scientific kits are used to reduce the risk of reagent contamination and improve detection accuracy.

[0021] In some embodiments, the samples prepared in steps S1-S8 need to be lysed before being sent to the fluorescent quantitative PCR instrument, and the lysing treatment includes:

[0022] S081: taking a predetermined volume of the sample, placing it in the fluorescent quantitative PCR instrument, and setting the temperature of the hot cover to 105° C.;

[0023] S082: Setting the first cracking program, the temperature is 50-80°C, and the duration is 10-25 minutes;

[0024] S083: setting the second cracking program, the temperature is 70-120°C, and the duration is 1-9 minutes;

[0025] S084: Repeat steps S082 and S083 1-3 times; and

[0026] S085: Set the third cracking program at a temperature of 0-5°C and store for later use.

[0027] The samples were lysed under the above conditions to provide a suitable environment for proteinase K to better peel off the proteins adhered to the DNA molecules and extract DNA more efficiently.

[0028] In some embodiments, after the lysis treatment is completed, the sample prepared in steps S1-S8 needs to be added with a sample detection treatment reagent and then sent to the fluorescent quantitative PCR instrument for detection. The sample detection procedure of the fluorescent quantitative PCR instrument is:

[0029] S091: digestion, temperature 30-80°C, duration 1-9 minutes;

[0030] S092: pre-denaturation, temperature is 95°C, duration is 10 minutes;

[0031] S093: Repeat the above steps S091 and S092 1-3 times;

[0032] S094: denaturation, temperature 70-120°C, duration 1-10 seconds;

[0033] S095: annealing / extension, temperature 40-80°C, duration 10-60 seconds, collect Ct value; and

[0034] S096: Repeat the above steps S094 and S095 20-60 times;

[0035] The temperature of the hot cover of the fluorescence quantitative PCR instrument is 105°C, the temperature change rate is 1.6°C / s, and the injection volume is 10-30 μl.

[0036] Through real-time sampling, the concentration of DNA molecules can be monitored in real time during the entire PCR amplification process, allowing the target sequence to be detected at very low DNA concentrations, greatly reducing the risk of false positive results. At the same time, analysis is performed while the sample reaction is growing exponentially, improving data accuracy and repeatability, shortening the time required for fluorescence detection of a single sample, and improving sample detection efficiency.

[0037] In some embodiments, the sample detection and processing reagents are from the Human Residual DNA (qPCR) Detection Kit with the product number HM0311C-100 produced by Shanxi Junyan, which is convenient to use and reduces the introduction of pollutants.

[0038] In some embodiments, the calibration samples are set to six groups, which are arranged from high to low according to the DNA molecule concentration as STD0, STD1, STD2, STD3, STD4, and STD5, wherein the DNA molecule concentration of the STD4 sample is at the same order of magnitude as that of the protein stock solution sample, and the DNA molecule concentration of the STD1 sample is at the same order of magnitude as that of the intracellular protein sample. By controlling the DNA molecule concentration of the STD4 sample to be at the same order of magnitude as that of the protein stock solution sample, and the DNA molecule concentration of the STD1 sample to be at the same order of magnitude as that of the intracellular protein sample, the compatibility of the standard curve prepared by the calibration sample with the protein polymer product to be detected is improved, thereby ensuring that the Ct value of the sample can fall within the standard curve, and the DNA molecule concentration can be smoothly converted.

[0039] In some embodiments, the DNA standard substance solution is prepared by diluting a DNA quantitative reference substance of a predetermined concentration;

[0040] The STD0 sample is prepared by adding the diluent solvent to the DNA quantitative reference;

[0041] The STD1 sample is prepared by adding the dilution solvent to the STD0 sample;

[0042] The STD2 sample is prepared by adding the dilution solvent to the STD1 sample;

[0043] The STD3 sample is prepared by adding the dilution solvent to the STD2 sample;

[0044] The STD4 sample is prepared by adding the dilution solvent to the STD3 sample;

[0045] The STD 5 sample is prepared by adding the dilution solvent to the STD 4 sample.

[0046] In some embodiments, the sample pretreatment agents are divided into multiple types, and the concentration of proteinase K in each sample pretreatment agent is different. The samples in steps S1-S8 are treated with multiple sample pretreatment agents respectively to form a control experiment of multiple groups of different concentration systems of proteinase K. By treating the above-mentioned samples with the concentration of proteinase K as a variable, it is intended to evaluate the effects of different concentrations of proteinase K on the extraction degree of DNA molecules in the above-mentioned samples and the effects on the detection results under the same lysis procedure and time conditions for protein stock solution samples and intracellular protein samples that may have different DNA molecule concentrations.

[0047] In some embodiments, the sample pretreatment agent is divided into two types, each of which is composed of a mixture of proteinase K and 10×Lysis Buffer, wherein the proteinase K concentration is divided into 1 / 10PK and 1 / 5PK, and the composition of the 1 / 10PK concentration system is: the volume ratio of proteinase K to 10×Lysis Buffer is 1:9; the composition of the 1 / 5PK concentration system is: the volume ratio of proteinase K to 10×Lysis Buffer is 1:4. The proteinase K content of the 1 / 5PK concentration system is twice the proteinase K content of the 1 / 10PK concentration system. The two concentration systems may have different DNA molecule extraction efficiencies for the same sample, which is convenient for screening out a proteinase K concentration that is more suitable for the current sample.

[0048] In some embodiments, each group of samples is set up with multiple replicate wells for experiment, so as to ensure the accuracy and repeatability of the results and reduce the impact of accidental errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a flowchart of the steps of an optional embodiment of the method for detecting residual host cell DNA of protein polymers of the present application.

[0050] Figure 2 It is a flow chart of the steps of the cleavage reaction after the sample is sent into a fluorescent quantitative PCR instrument in an optional embodiment of the host cell DNA residue detection method of the protein polymer of the present application.

[0051] Figure 3 This is a flow chart of the steps of sending a sample into a fluorescent quantitative PCR instrument for real-time detection in an optional embodiment of the method for detecting residual host cell DNA of a protein polymer of the present application. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further explained below in conjunction with experiments.

[0053] The production process of intracellular protein products of host cells used in the following experiments includes:

[0054] Cultivating mesenchymal stem cells and using ultraviolet irradiation to create a stress environment;

[0055] After irradiation, cells were harvested, the culture was filtered through a 300-mesh filter bag to retain microcarriers, and the supernatant was centrifuged at 1200 rpm for 6 minutes. It was stored at 4°C, the cell pellet was taken, washed with physiological saline three times, and finally pure water was added to repeatedly blow and lyse the cells, and then filtered through a 0.22 μm filter membrane to harvest the intracellular protein product.

[0056] The production process of the protein polymer stock solution used in the following experiments includes:

[0057] The above-mentioned intracellular protein product was taken and separated and purified by size exclusion chromatography to obtain a protein polymer stock solution product; wherein, under the size exclusion chromatography condition of a flow rate of 0.2 ml / min, the elution volume of the first component peak was 12-13.2 ml, the elution volume of the second component peak was 15.2-17 ml, the elution volume of the third component peak was 17-20 ml, the elution volume of the fourth component peak was 29-31 ml, and the elution volume of the fifth component peak was 31-34 ml.

[0058] A method for detecting the residual amount of host cell DNA in a protein polymer is performed by using a fluorescent quantitative PCR instrument on a protein stock solution sample and an intracellular protein sample. The method comprises:

[0060] S1: Add dilution solvent to DNA standard substance to prepare several groups of calibration samples with different DNA molecular concentrations, and send them into fluorescent quantitative PCR instrument for detection to form a standard curve correlating DNA molecular concentration and Ct value;

[0062] S2: adding a sample pretreatment agent into the protein polymer stock solution product to prepare a protein stock solution sample;

[0063] S3: adding a sample pretreatment agent and the calibration sample into the protein polymer stock solution product to prepare a protein stock solution spiked sample;

[0065] S4: adding a sample pretreatment agent into the intracellular protein product of the host cell of the protein polymer to prepare an intracellular protein sample;

[0067] S5: adding a sample pretreatment agent and the calibration sample into the intracellular protein product of the host cell of the protein polymer to prepare an intracellular protein spiked sample;

[0069] S6: Design a blank sample, wherein the blank sample is the dilution solvent;

[0070] S7: Design a negative control sample, wherein the negative control sample is the sample pretreatment agent;

[0071] S8: designing a positive control sample, wherein the positive control sample is prepared by mixing the calibration sample with the sample pretreatment agent;

[0073] S9: sending the sample obtained in steps S2-S8 into a fluorescent quantitative PCR instrument for detection to obtain a Ct value; and

[0074] S10: evaluating and judging the credibility of the Ct values ​​measured in step S9, and substituting the credible Ct values ​​into the standard curve one by one to obtain the corresponding DNA molecule concentration of the sample;

[0075] The evaluation and judgment of the Ct value includes: comparing the Ct values ​​of the negative control sample, the blank sample and the calibration sample with the lowest concentration; when the Ct values ​​of the negative control sample and the blank sample are greater than the Ct value of the DNA standard substance solution with the lowest concentration, it is determined that the Ct values ​​determined in step S9 are credible; when at least one of the Ct values ​​of the negative control sample and the blank sample is less than the Ct value of the calibration sample with the lowest concentration, it is determined that the Ct value determined in step S9 is not credible, and the parameters need to be adjusted to repeat the above steps S1-S9.

[0076] Among them, the main instruments and equipment

[0077]

[0078] Main reagents and consumables

[0079]

[0080]

[0081] In addition, it should be noted that the above-mentioned Human residual DNA (qPCR) detection kit contains 2×qPCR Reaction Buffer and Human Primer&Probe MIX, and the above-mentioned 10× cell / virus lysis kit contains 10× Lysis Buffer and proteinase K.

[0082] Sample pretreatment agent preparation:

[0083] Vortex and mix 10× Lysis Buffer and proteinase K in a ratio of 9:1, centrifuge quickly to obtain a sample pretreatment agent containing 1 / 10 PK, leave it at room temperature, and take it out as needed;

[0084] Vortex mix 10× Lysis Buffer and proteinase K in a ratio of 4:1, quickly centrifuge and obtain a sample pretreatment agent containing 1 / 5 PK. Leave it at room temperature and take it out when needed.

[0085] Preparation of calibration samples and blank samples:

[0086] A DNA quantitative reference with a known DNA molecular concentration of 30000 ng / ml was used and diluted according to Table 1 to obtain STD0-STD5 calibration samples and blank samples:

[0087] Table 1

[0088] Calibration sample name Dilution process Concentration (ng / ml) STD0 10μl DNA quantitative reference + 90μl DNA dilution solution 3000 STD1 10μl STD0 + 90μl DNA dilution buffer 300 STD2 10μl STD1 + 90μl DNA dilution buffer 30 STD3 10μl STD2 + 90μl DNA dilution buffer 3 STD4 10μl STD3 + 90μl DNA dilution buffer 0.3 STD5 10μl STD4 + 90μl DNA dilution buffer 0.03 blank solution 100 μl DNA dilution buffer 0

[0089] Negative control sample preparation:

[0090] Prepare negative control samples according to Table 2:

[0091] Table 2

[0092] Sample name Preparation process Negative control-1 PCR diluent + 1 / 10 PK sample pretreatment agent, mixed Negative control-2 PCR diluent + 1 / 5 PK sample pretreatment agent, mixed

[0093] Positive control sample preparation:

[0094] Prepare the positive control sample according to Table 3:

[0095] Table 3

[0096] Sample name Preparation process Positive control-1 STD4 sample + 1 / 10PK sample pretreatment agent, mixed Positive Control-2 STD1 sample + 1 / 10PK sample pretreatment agent, mixed Positive Control-3 STD4 sample + 1 / 5PK sample pretreatment agent, mixed Positive Control-4 STD1 sample + 1 / 5PK sample pretreatment agent, mixed

[0097] Preparation of protein stock sample:

[0098] Prepare the protein stock solution sample according to Table 4:

[0099] Table 4

[0100] Sample name Preparation process Protein stock solution sample-1 Protein stock solution + 1 / 10PK sample pretreatment agent, mixed Protein stock solution sample-2 Protein stock solution + 1 / 5PK sample pretreatment agent, mixed

[0101] Prepare protein stock spike samples:

[0102] Prepare protein stock solution spiked samples according to Table 5:

[0103] Table 5

[0104] Sample name Preparation process Protein stock solution spike-1 Protein stock solution + STD2 sample + 1 / 10PK sample pretreatment agent, mixed Protein stock solution spike-2 Protein stock solution + STD3 sample + 1 / 10PK sample pretreatment agent, mixed Protein stock solution spike-3 Protein stock solution + STD4 sample + 1 / 10PK sample pretreatment agent, mixed Protein stock solution spike-4 Protein stock solution + STD2 sample + 1 / 5PK sample pretreatment agent, mixed Protein stock solution spike-5 Protein stock solution + STD3 sample + 1 / 5PK sample pretreatment agent, mixed Protein stock solution spike-6 Protein stock solution + STD4 sample + 1 / 5PK sample pretreatment agent, mixed

[0105] Preparation of intracellular protein samples:

[0106] Prepare intracellular protein samples according to Table 6:

[0107] Table 6

[0108]

[0109] Preparation of intracellular protein spike samples:

[0110] Prepare the intracellular protein spike samples according to Table 7:

[0111] Table 7

[0112]

[0113] The above samples are placed in a PCR instrument for cleavage reaction, and the cleavage treatment steps are as follows:

[0114] S081: taking a predetermined volume of the sample, placing it in the fluorescent quantitative PCR instrument, and setting the temperature of the hot cover to 105° C.;

[0115] S082: Setting the first cracking program, the temperature is 50-80°C, and the duration is 10-25 minutes;

[0116] S083: setting the second cracking program, the temperature is 70-120°C, and the duration is 1-9 minutes;

[0117] S084: Repeat steps S082 and S083 1-3 times; and

[0118] S085: Set the third cracking program at a temperature of 0-5°C and store for later use.

[0119] After the above lysis treatment operation was performed on each group of samples in Tables 1 to 7, PCR diluent was added for dilution.

[0120] Preparation before PCR real-time detection:

[0121] The reagents in the Human Residual DNA (qPCR) Detection Kit were thawed at room temperature, and 2×qPCR Reaction Buffer and Human Primer&Probe MIX were vortexed and mixed at a ratio of 3:1, and then centrifuged quickly to obtain the PCR reaction solution for later use.

[0122] Take a fluorescent quantitative PCR plate, add 20 μl of PCR reaction solution to each well, then take 10.00 μl of the lysed and diluted samples in Tables 1 to 7 above and add them to the corresponding wells. Set up 2 replicate wells for each sample, then seal the PCR plate, vortex mix, centrifuge in a compound rotor centrifuge for 1 minute, and finally put it into a fluorescent quantitative PCR instrument, set the hot cover temperature to 105°C, the temperature change speed to 1.6°C / s, the injection volume to 15 μl, and the detection program is as follows:

[0123] S091:

[0124] Digestion, temperature set at 30-80°C, duration 1-9 minutes;

[0125] S092: pre-denaturation, temperature set to 70-120°C, duration for 10 minutes;

[0126] S093: Repeat the above steps S091 and S092 1-3 times;

[0127] S094: denaturation, temperature set to 70-120°C, duration 1-10 seconds;

[0128] S095: annealing / extension, the temperature is set to 40-80°C, the duration is 10-60 seconds, and the Ct value is collected; and

[0129] S096: Repeat the above steps S094 and S095 20-60 times;

[0130] The temperature of the hot cover of the fluorescence quantitative PCR instrument is 105°C, the temperature change rate is 1.6°C / s, and the injection volume is 10-30 μl.

[0131] The built-in software of the fluorescence quantitative PCR instrument will automatically report the mean value (Mean Qty), standard deviation (StdDev Qty), Ct standard deviation (StDev Ct), Ct standard deviation (StDev Ct) of Standard replicate wells, and the linear determination coefficient R of the standard curve. 2 , slope (Slope).

[0132] Statistical treatment:

[0133] Relative standard deviation (RSD), RSD = (StdDev Qty) / (Mean Qty) × 100%;

[0134] The residual amount of host cell DNA, Human HCD residual (ng / ml) = Q × F, Q is the qPCR detection value, and F is the equivalent dilution multiple.

[0135] Experimental results analysis

[0136] The experimental results of cleavage using 1 / 10 proteinase K are shown in Tables 8 and 9:

[0137] Table 8

[0138]

[0139] The Ct values ​​of the blank sample and the negative control-1 sample are greater than the Ct value of the STD5 sample, which means that the blank sample containing only DNA diluent and the negative control-1 sample containing only PCR diluent, proteinase K and 10×Lysis Buffer will not introduce additional DNA molecular impurities exceeding the detection limit of the current instrument. Therefore, it can be determined that the samples prepared by the methods of Tables 1 to 7 above are not introduced with contaminants that affect the detection values, and therefore the measured detection values ​​are credible.

[0140] Table 9

[0141]

[0142]

[0143] in conclusion:

[0144] The detection values ​​of the protein stock solution-1 sample and the protein stock solution spiked-3 sample were lower than the lower limit of the standard curve, so the measured DNA molecular concentration values ​​and RSD were for reference only;

[0145] The RSD of samples with detection values ​​within the standard curve range was 1.1% to 11.3%, which met the credible standard; the recovery rate of positive control samples was 116.54% to 129.04%, which met the credible standard;

[0146] For protein stock samples, the recovery rate of protein stock spiked samples within the standard curve range was 98.85% to 106.99%, which met the credible standard; for intracellular protein samples, the recovery rate of intracellular protein spiked samples within the standard curve range was 156.49%, which did not meet the standard. Therefore, the pre-treatment method using 1 / 10 proteinase K for cleavage is suitable for the detection of host cell DNA residues in protein stock products, but not for the detection of host cell DNA residues in intracellular protein products.

[0147] It is understandable that the above experiment can be repeated by adjusting the concentration parameters of proteinase K used to treat the sample, the concentration parameters of the calibrated sample, or the dilution multiple parameters before the sample test so that the recovery rate of the intracellular protein spiked sample meets the credible standard.

[0148] The experimental results of cleavage using 1 / 5 proteinase K are shown in Tables 10 and 11:

[0149] Table 10

[0150]

[0151] The Ct values ​​of the blank sample and the negative control-2 sample are greater than the Ct value of the STD5 sample, which means that the blank sample containing only DNA diluent and the negative control-2 sample containing only PCR diluent, proteinase K and 10×Lysis Buffer will not introduce additional DNA molecules and / or other impurity molecules that exceed the detection limit of the current instrument. Therefore, it can be determined that the samples prepared by the methods of Tables 1 to 7 above are not introduced with contaminants that affect the detection values, and therefore the measured detection values ​​are credible.

[0152] Table 11

[0153]

[0154] in conclusion:

[0155] The detection values ​​of protein stock solution-2 sample and protein stock solution spiked-6 sample were lower than the lower limit of the standard curve, so the measured DNA molecular concentration value and RSD were for reference only; the recovery rate of the positive control sample was 146.51% to 153.61%, which did not meet the credible standard; for protein stock solution spiked samples, the recovery rate of spiked samples within the standard curve range was 116.80% to 128.12%, which met the credible standard; for intracellular protein samples, the recovery rate of spiked samples within the standard curve range was 175.90%, which did not meet the credible standard. Therefore, the pre-treatment method using 1 / 5 proteinase K for cleavage is not suitable for the detection of host cell DNA residues in protein stock products and intracellular protein products.

[0156] It is understandable that the concentration parameters of proteinase K used to treat the sample, the concentration parameters of the calibrated sample, or the dilution multiple parameters before the sample test can be further adjusted to repeat the above experiment so that the recovery rate of each of the above samples meets the credible standard.

[0157] By verifying the credibility of the DNA molecule concentration measured by the above-mentioned DNA residue detection method, the average value and relative standard deviation of the detection value of the DNA molecule concentration of each sample are calculated respectively, and the recovery rate of the sample with the addition of the calibration sample compared with the sample without the addition of the calibration sample is calculated, the samples with the detection value of the DNA molecule concentration greater than or equal to the minimum value of the standard curve, the relative standard deviation ≤30% and the recovery rate of 50%-150% are screened out and used as credible samples with credible detection values, so as to achieve accurate detection of the host cell DNA residue of the protein polymer, and provide data support for judging the purity and safety of use of the protein polymer product.

[0158] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for detecting residual host cell DNA of a protein polymer, characterized in that: The method comprises: S1: Add dilution solvent to DNA standard substance to prepare several groups of calibration samples with different DNA molecular concentrations, and send them into fluorescent quantitative PCR instrument for detection to form a standard curve correlating DNA molecular concentration and Ct value; S2: adding a sample pretreatment agent into the protein polymer stock solution product to prepare a protein stock solution sample; S3: adding a sample pretreatment agent and the calibration sample into the protein polymer stock solution product to prepare a protein stock solution spiked sample; S4: adding a sample pretreatment agent into the intracellular protein product of the host cell of the protein polymer to prepare an intracellular protein sample; S5: adding a sample pretreatment agent and the calibration sample into the intracellular protein product of the host cell of the protein polymer to prepare an intracellular protein spiked sample; S6: Design a blank sample, wherein the blank sample is the dilution solvent; S7: Design a negative control sample, wherein the negative control sample is the sample pretreatment agent; S8: designing a positive control sample, wherein the positive control sample is prepared by mixing the calibration sample with the sample pretreatment agent; S9: sending the sample obtained in steps S2-S8 into a fluorescent quantitative PCR instrument for detection to obtain a Ct value; and S10: evaluating and judging the credibility of the Ct values ​​measured in step S9, and substituting the credible Ct values ​​into the standard curve one by one to obtain the corresponding DNA molecule concentration of the sample; The evaluation and judgment of the Ct value includes: comparing the Ct values ​​of the negative control sample, the blank sample and the calibration sample with the lowest concentration; when the Ct values ​​of the negative control sample and the blank sample are greater than the Ct value of the DNA standard substance solution with the lowest concentration, it is determined that the Ct values ​​determined in step S9 are credible; when at least one of the Ct values ​​of the negative control sample and the blank sample is less than the Ct value of the calibration sample with the lowest concentration, it is determined that the Ct value determined in step S9 is not credible, and the parameters need to be adjusted to repeat the above steps S1-S9.

2. The method for detecting residual DNA according to claim 1, characterized in that: The fluorescent quantitative PCR instrument is a fluorescent quantitative PCR instrument of model Quant Studio 5 produced by ABI, the dilution solvent is a DNA diluent, and the sample pretreatment agent is a 10× cell / virus lysis kit with product number AN19L050 produced by Heyuan Liji (Shanghai) Biotechnology Co., Ltd.

3. The method for detecting residual DNA according to claim 2, characterized in that: The samples prepared in steps S1-S8 need to be lysed before the fluorescence quantitative detection, and the lysing process includes: S081: taking a predetermined volume of the sample, placing it in the fluorescent quantitative PCR instrument, and setting the temperature of the hot cover to 105° C.; S082: setting the first cracking program, the temperature is 50-80°C, and the duration is 10-25 minutes; S083: setting the second cracking program, the temperature is 70-120°C, and the duration is 1-9 minutes; S084: Repeat steps S082 and S083 1-3 times; and S085: Set the third cracking program at a temperature of 0-5°C and store for later use.

4. The method for detecting residual DNA according to claim 3, characterized in that: After the lysis treatment is completed, the sample prepared in steps S1-S8 needs to be added with a sample detection treatment reagent and then sent to the fluorescent quantitative PCR instrument for detection. The sample detection procedure of the fluorescent quantitative PCR instrument is as follows: S091: digestion, temperature set to 30-80°C, duration 1-9 minutes; S092: pre-denaturation, temperature set to 70-120°C, duration for 10 minutes; S093: Repeat the above steps S091 and S092 1-3 times; S094: denaturation, temperature set to 70-120°C, duration 1-10 seconds; S095: annealing / extension, the temperature is set to 40-80°C, the duration is 10-60 seconds, and the Ct value is collected; and S096: Repeat the above steps S094 and S095 20-60 times; The temperature of the hot cover of the fluorescence quantitative PCR instrument is 105°C, the temperature change rate is 1.6°C / s, and the injection volume is 10-30 μl.

5. The method for detecting residual DNA according to claim 4, characterized in that: The sample detection and processing reagents come from the Human residual DNA (qPCR) detection kit with the product number HM0311C-100 produced by Shanxi Junyan.

6. The method for detecting residual DNA according to claim 1, characterized in that: The calibration samples are set to six groups, which are sorted from high to low in terms of DNA molecule concentration as STD0, STD1, STD2, STD3, STD4, and STD5, wherein the DNA molecule concentration of the STD4 sample is at the same order of magnitude as that of the protein stock solution sample, and the DNA molecule concentration of the STD1 sample is at the same order of magnitude as that of the intracellular protein sample.

7. The method for detecting residual DNA according to claim 6, characterized in that: The DNA standard substance solution is prepared by diluting a DNA quantitative reference substance of a predetermined concentration; The STD0 sample is prepared by adding the diluent solvent to the DNA quantitative reference; The STD1 sample is prepared by adding the dilution solvent to the STD0 sample; The STD2 sample is prepared by adding the dilution solvent to the STD1 sample; The STD3 sample is prepared by adding the dilution solvent to the STD2 sample; The STD4 sample is prepared by adding the dilution solvent to the STD3 sample; The STD 5 sample is prepared by adding the dilution solvent to the STD 4 sample.

8. The method for detecting residual DNA according to claim 2, characterized in that: There are multiple types of sample pretreatment agents, each of which has a different concentration of proteinase K. The samples in steps S1-S8 are treated with multiple types of sample pretreatment agents, respectively, to form multiple groups of control experiments with different proteinase K concentration systems.

9. The method for detecting residual DNA according to claim 8, characterized in that: The sample pretreatment agent is divided into two types, each of which is composed of a mixture of proteinase K and 10×Lysis Buffer, wherein the proteinase K concentration is divided into 1 / 10PK and 1 / 5PK, and the composition of the 1 / 10PK concentration system is: the volume ratio of proteinase K to 10×Lysis Buffer is 1:9; the composition of the 1 / 5PK concentration system is: the volume ratio of proteinase K to 10×Lysis Buffer is 1:

4.

10. The method for detecting residual DNA according to claim 1, characterized in that: Multiple replicate wells were set up for each group of samples for experiment.

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