Plasmid target spot, primer probe, kit and method for detecting host DNA residue in cell preparation or virus preparation

Abstract

The invention relates to a plasmid target spot, a primer probe, a kit and a method for detecting host DNA residues in a cell preparation or a virus preparation. The invention relates to the technical field of biology, in particular to a plasmid target spot, a primer probe, a kit and a method for detecting host DNA residues in a cell preparation or a virus preparation. The plasmid target point is selected from one or two of plasmid Ori and KanR genes, the Ori element has a gene sequence as shown in SEQ ID NO.1, the KanR gene has a gene sequence capable of coding a Kan R peptide chain, such as a nucleotide sequence for coding SEQ ID NO.2, the nucleotide sequence can have any one nucleotide sequence in SEQ ID NO.3-5, and the Kan R has an amino acid sequence as shown in SEQ ID NO.2. The invention also relates to a primer probe of the plasmid target, a kit and a use method. The invention provides an effective detection target (Ori and KanR genes) and an accurate detection amplification system, and detection of host DNA residues and plasmid DNA residues in a cell preparation or a virus preparation is realized.

Description

[0001] This application is a divisional application. The original application was filed on October 26, 2022, with application number 202280005530.8 (PCT / CN2022 / 127776), and the invention title was "Plasmid target, primer probe, kit and method for detecting host DNA residues in cell preparations or viral preparations". Technical Field

[0002] This application relates to the field of biotechnology, specifically to plasmid targets, primers, probes, kits, and methods for detecting residual host DNA in cell preparations or viral preparations. Background Technology

[0003] Cell and gene therapy has become the hottest sector in biopharmaceuticals after antibody drugs. According to FDA statistics, the number of cell and gene therapies entering early-stage clinical development has surged recently, with an estimated 200 or more IND applications received annually. As of December 6, 2021, the FDA had approved 22 cell and gene therapy products.

[0004] Various viral vectors are used in cell and gene therapy, including adenoviruses, lentiviruses, and adeno-associated viruses. These vectors are mostly produced using HEK293T cells, and residual DNA can exist as an impurity in the final product. Regulatory agencies in various countries have set limits on the amount of residual host DNA. For example, the 2020 edition of the Chinese Pharmacopoeia states that the residual amount of process-related impurities such as host cell DNA should be tested and controlled within acceptable levels [General Principles of Human Gene Therapy Products; Chinese Pharmacopoeia (Part III) 2020 Edition]. The "Technical Guidelines for Research and Evaluation of Gene Transduction and Modification Systems Pharmacy (Draft for Comments)" released in 2020 recommends that applicants set the limit for residual DNA from non-tumor-causing cells at less than 10 ng / dose.

[0005] In viral-vectored CAR-T and TCR-T cell therapies, both the finished cell product and HEK293T contain human DNA, and the proportion of residual host DNA (HCD) relative to the total DNA in the finished cell product is low, making accurate detection difficult. Current methods still rely on detecting residual host DNA in the viral preparation, strictly controlling the HCD level in the viral preparation to ensure that the HCD in the cell preparation does not exceed the limit. However, overly strict control of HCD in viral preparations makes the process exploration cumbersome and costly.

[0006] Therefore, there is still a need in this field for a means to accurately and conveniently detect residual host DNA in cell preparations. Summary of the Invention

[0007] In view of this, the present invention provides a method for detecting residual host DNA in finished cell products. In this invention, the residual host DNA in the finished cell product is used to optimize related processes, reduce excessive process requirements, and ultimately lower costs. Specifically, according to the method of the present invention, residual host DNA (R1) is first detected in the viral preparation. The residual plasmid in the viral preparation (P1) and the residual plasmid in the cell preparation (P2) are then detected to calculate the extracellular DNA clearance rate during cell culture, thereby calculating the residual host DNA in the cell preparation: R2 = R1 * (P2 / P1).

[0008] In a first aspect, this application provides a plasmid target for detecting residual host DNA in cell preparations or viral preparations, said plasmid target being selected from one or both of the plasmid Ori element and the KanR gene. In some embodiments, the Ori element has the gene sequence of SEQ ID NO.1, and the KanR gene has a gene sequence capable of encoding a Kan R peptide chain, such as the nucleic acid sequence encoding SEQ ID NO.2. In some further embodiments, the KanR gene is a gene sequence encoding the amino acid sequence of SEQ ID NO.2, such as a gene sequence having any one of SEQ ID NO.3-5. In some other embodiments, in addition to the gene sequence disclosed herein, the KanR gene may also be other derived sequences capable of encoding a Kan R peptide chain, the similarity of which to the gene sequence disclosed herein is maintained at 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more. In some embodiments, plasmids containing the Ori element can be used as vectors, such as PUC19, PUC18, PMD18-T, etc. In some implementations, the aforementioned primers and probes can be fluorescently labeled, such as 5'HEX, 3'BHQ1; 5'VIC, 3'BHQ1; 5'TAMRA, 3'BHQ2; 5'ROX, 3'BHQ3; 5'CY5, 3'BHQ2 or 5'CY5, 3'BHQ3, etc.

[0009] In some embodiments, the cell preparation is selected from TCR-T, CAR-T, CAR-NK and CAR-M cell preparations, and the viral preparation is selected from lentivirus and adenovirus.

[0010] In a second aspect, this application provides a primer probe according to the first aspect for detecting plasmid target sites for host DNA residues in cell preparations or viral preparations, wherein for the plasmid Ori element, the primer probe is selected from one of SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8; and for the plasmid KanR gene, the primer probe is selected from one of SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11.

[0011] In a third aspect, this application provides a kit for detecting residual host DNA in cellular or viral preparations, comprising the primers and probes described in the second aspect.

[0012] In a fourth aspect, this application provides a method for detecting host DNA residues in cell preparations or viral preparations, which may include the following steps: detecting host DNA residues in viral preparations (R1), detecting plasmid residues in viral preparations (P1) using the primer probes described in the second aspect, and detecting plasmid residues in cell preparations (P2) to calculate the extracellular DNA clearance rate during cell culture, and then calculating the host DNA residues in the cell preparations according to the following formula: R2 = R1 * (P2 / P1).

[0013] In some implementations, residual DNA detection kits are used to detect residual host DNA in viral preparations (R1).

[0014] In some embodiments, detecting plasmid residues (P1) in a viral preparation using the primer probes described in the second aspect includes the following steps: 1) primer design; 2) preparing quantitative standards containing Ori and / or KanR elements using a plasmid vector; 3) configuring primer probes containing Ori or KanR elements into a primer mixture, adding them to an amplification mixture for amplification, and obtaining an amplification standard curve; and 4) calculating plasmid residues (P1) in the viral preparation based on the amplification standard curve.

[0015] In some implementations, primers using Ori or KanR elements are used to detect plasmid residues in cell preparations.

[0016] In summary, this application can achieve at least one of the following beneficial technical effects: This invention provides a method for detecting residual host DNA in finished cell therapy products. The method detects plasmid residues in both viral vectors and finished cell products, and calculates the residual DNA clearance rate during the process from viral vector to finished cell product based on the ratio of these two residues. Based on the DNA clearance rate and the detection of host DNA content in the viral vector, the residual host DNA in the finished cell product is calculated. This method enables the detection of residual host DNA in finished cell products, solving the problem of large discrepancies between detected values ​​and actual values ​​when detecting host residues at the viral vector end.

[0017] It provides a method for detecting plasmid residues, offers effective detection targets (Ori and KanR elements), and provides an accurate detection amplification system. Attached Figure Description

[0018] Figure 1 This is a general process flow diagram for the production of CAR-T and TCR-T cell therapy formulations in existing technologies; Figure 2 This is the standard curve amplification diagram of the Ori primer system in Example 1; Figure 3 This is the standard curve amplification diagram of the KanR primer system in Example 1; Figure 4-8 The results are as follows: Flow cytometry analysis of Jurkat cells infected with the virus, diluted 20, 540, 1620, and 4860 times with 100 μL added in Example 2. Figure 9 The results of plasmid residue detection for virus diluted 10, 100, 1000, 10000, and 100000 times in Example 2 are as follows; Figure 10 This is a residual amplification diagram of the plasmid in the mouse PBMC cell preparation from Example 2; Figure 11 The flow cytometry results of the mouse PBMC cell preparation from Example 3 are shown. Figure 12 This is an amplification diagram of plasmid residue detection of viral vector using KanR primers in Example 3; Figure 13 Example 3 shows the amplification diagram of residual plasmids in the finished cell product detected by the KanR primer system; Figure 14 Example 4 shows the amplification diagram of residual lentiviral plasmid detection using Ori primers; Figure 15 This is Example 4, showing the amplification diagram of residual lentiviral plasmid detection using Kan primers; Figure 16 This is the flow cytometry result of the CD3 cell preparation in Example 4; Figure 17Example 4 shows the amplification diagram of residual plasmids in the finished cell product detected by the Ori primer system; Figure 18 This is a diagram from Example 4 showing the detection of residual plasmid amplification in the finished cell product using the KanR primer system. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, those skilled in the art will understand that the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0020] The main steps in producing CAR-T and TCR-T cell therapy formulations include initial isolation and enrichment of T cells, T cell activation, CAR / TCR gene transfer using viral or non-viral vector systems, in vitro T cell expansion, and final end-processing and cryopreservation. See details below. Figure 1 The process flow diagram is shown below. In this process, the transformation from viral vector to finished cell product involves cell medium exchange or host cell DNA degradation during cell culture. The residual host DNA in the virus is higher than in the finished cell product, therefore the detected value cannot reflect the actual residual host DNA. The pharmacopoeia only specifies requirements for the residual host DNA in the finished product; when the residual value in the viral vector exceeds the pharmacopoeia requirement, the host DNA content must be reduced in the viral process. Reducing the residual host DNA in the viral vector requires complex process exploration, such as adding DNase for degradation. However, excessive DNase addition can lead to excessively high DNase residues and increase the difficulty of subsequent viral vector purification processes, posing significant challenges to process requirements and costs. Simultaneously, the HCD value must meet the requirements for cell preparation during the viral formulation process, which is overly stringent because residual DNA is removed during transduction and cell culture through medium exchange and other operations.

[0021] Therefore, if a detection method can be developed to accurately calculate the clearance rate, the accurate HCD value in cell preparations can be obtained. This is of great value for evaluating whether cell preparations meet pharmacopoeia requirements, guiding the exploration of virus processes, and formulating virus quality release standards.

[0022] The inventors of this application conducted extensive research on this matter. During the research, the inventors realized that the finished cell preparation, TCR T, is human-derived, and the residual viral host DNA is also human-derived. Therefore, it is impossible to distinguish whether the DNA extracted from the cell preparation is residual host DNA introduced by the addition of the virus or originates from the TCR T cell preparation itself. Simultaneously, the inventors discovered that residual plasmids and residual host DNA in both the virus and the finished cell preparation exhibit the same reduction ratio, i.e., clearance rate, during the process from viral vector to finished cell preparation. Therefore, by detecting plasmid residues in the finished cell preparation and the viral vector, and comparing the ratio of plasmid residues in both, the clearance rate of residual host DNA from the cell process can be calculated. Then, based on the amount of residual host DNA in the viral vector and the DNA clearance rate, the amount of residual host DNA in the finished cell preparation can be determined, as illustrated below. Plasmids and host DNA have the same DNA clearance rate during cell processing, meaning that from the viral vector to the final cell product, plasmid residue and host DNA residue have the same remaining proportion. The calculation formula is: Host DNA residue = Plasmid residue in the final cell product / Plasmid residue in the viral vector * Host DNA residue in the plasmid vector.

[0023] In other words, by detecting non-human plasmid residues during the viral phase and plasmid residues during the cell preparation phase to calculate residual DNA clearance efficiency, it is possible to indirectly calculate host DNA residues in cell preparations. Based on this, the inventors of this application completed the present invention.

[0024] Specifically, this invention provides a method for detecting residual host DNA in cell preparations. In this method, the residual host DNA in the finished cell product is used to optimize related processes, reduce excessive process requirements, and ultimately lower costs. First, residual host DNA (R1) is detected in the viral preparation. Then, the extracellular DNA clearance rate during cell culture is calculated by detecting plasmid residues in the viral preparation (P1) and in the cell preparation (P2). Finally, the residual host DNA in the cell preparation is calculated as follows: R2 = R1 * (P2 / P1). This method allows for rapid and accurate detection of the DNA residue rate in the cell preparation. Simultaneously, accurate detection of the residual host DNA in the finished product, while ensuring compliance with pharmacopoeia requirements, can better guide viral and cell processes.

[0025] The present invention will now be described in further detail with reference to the embodiments. Those skilled in the art will understand that these embodiments are given merely to facilitate understanding and implementation of the invention, and are not intended to limit the scope of protection of the invention to these specific embodiments.

[0026] Example 1: Detection of plasmid residues using Ori and KanR elements on the plasmid as targets. 1) Primer design As shown in Table 1, detection primers and probes were designed for Ori (SEQ ID NO.1) and KanR (SEQ ID NO.3), respectively: Table 1: Detection primers and probes for Ori (SEQ ID NO.1) and Kan R (SEQ ID NO.3) Primers were synthesized at Sangon Biotech (Shanghai) Co., Ltd., and then purified by HPLC.

[0027] 2) Preparation of quantitative standards The KanR (SEQ ID NO.3) sequence was ligated into a PUC57 vector carrying the Ori element to prepare quantitative standards containing Ori and Kan targets. Plasmids were extracted and their concentration was measured using a Nanodrop 2000 (Thermofisher, USA). 1 μg of plasmid was linearized by restriction endonuclease QuickCut™ EcoRI (Takara Bio Engineering (Dalian) Co., Ltd., catalog number 1611). The plasmid concentration was diluted to 3E8 copies / μl according to the amount of restriction enzyme added and the plasmid molecular weight. The plasmid standards were then aliquoted into 100 μl vials.

[0028] 3) qPCR amplification system and detection Primers and probes for the Ori or KanR genes were prepared as a 20x Primer MIX mixture. The concentrations of the upstream and downstream primers and probes in the mixture were 8 μM, 8 μM, and 4 μM, respectively.

[0029] The amplification mixture was Premix Ex Taq™ (Probe qPCR) (Takara Bio Engineering (Dalian) Co., Ltd., catalog number RR390A). 20 μL reaction mixtures were prepared using Ori and Kan primers, respectively; specific sample volumes are shown in Table 2.

[0030] Table 2: Amplification System Configuration Take 10 μL of the plasmid standard from step 2), add 90 μL of EASY Dilution (for Real Time PCR) (Takara Bio Engineering (Dalian) Co., Ltd., catalog number 9160) to dilute the plasmid standard to prepare a 3E7 copies / μL solution. Continue with a 10-fold serial dilution to prepare STD0, STD1, STD2, STD3, STD4, and STD5 concentrations of 3E6 copies / μL, 3E5 copies / μL, 3E4 copies / μL, 3E3 copies / μL, 3E2 copies / μL, and 30 copies / μL, respectively.

[0031] Dispense 17 μl into each well of a qPCR tube (96-well plate) and add 3 μl of each concentration standard. Perform the detection using a QTOWER3G real-time PCR instrument (Analytik Jena AG, Germany). The amplification program is shown in Table 3 below.

[0032] Table 3: Amplification Procedure *: Collect fluorescence signals (select the fluorescence channel corresponding to the probe). 4) Amplification results The amplification results are shown in Table 4 below. As can be seen from Table 4, the amplification efficiencies of both Ori and Kan primers are greater than 0.9, the linearity R² is greater than 0.98, and the difference between the theoretical values ​​and the calculated concentrations of the standards based on the standard curve is within 80%-120%. This indicates that the primers and standards amplified well.

[0033] Table 4: Amplification results of Ori and Kan primers The standard curve amplification graph of the Ori primer system is attached. Figure 2 The standard curve amplification diagram of the Kan primer system is attached. Figure 3 .

[0034] 5) Limit of Quantification The Ori and Kan primers were used to amplify the STD5 concentration plasmid. The amplification was repeated 20 times, and all amplifications were successful with a CV value of less than 10%, indicating that the limit of quantitation was less than 30 copies / ul.

[0035] Example 2 Method Validation: Comparison of the results of host DNA residue detection and plasmid residue calculation in mouse TCR-T finished products The purpose of providing this embodiment is to demonstrate that the method according to the present invention can accurately reflect the residual host DNA value in the cell preparation, proving the method's reliability and accuracy. The verification approach is as follows: Following the process for producing human TCR T cell preparations, but replacing the initial human PBMCs with mouse PBMCs, adding virus (accurately measuring residual host DNA (R1) and plasmid residue (P1)), and producing according to the same T cell activation, amplification, perfusion, and washing process, finally obtaining mouse TCR-T cells. At this point, the cell preparation can accurately measure both plasmid residue (P2) and human-derived residual host DNA (R2-TRUE). Then, the R2 calculated based on P2 / P1*R1=R2 is compared with the actually measured R2-TRUE.

[0036] 1. Production of mouse TCR using a human-mimicking TCR process: 1) Initial viral titer and R1 and P1 measurements HEK293T cells were co-transfected with a four-plasmid system (packaging plasmids pMD2.G (Addgene Plasmid #12259), pRSV-Rev (Addgene Plasmid #12253), and pMDLg / pRRE (Addgene Plasmid #12251), purchased from Addgene; and the transfer plasmid was a modified pLenti.PGK.chFP.W (Addgene Plasmid #51008) with the target TCR gene added) to package the virus. The purified virus was then used to infect Jurkat cells to detect the infection titer.

[0037] (1) Virus titer detection: Jurkat cells were at a density of 5 × 10^5 cells / mL, and 1 mL of cells were seeded into 24-well cell culture plates. Lentiviral cells were removed from -80 °C and thawed at 4 °C. After thawing, the cells were mixed and diluted with DMEM medium. 100 μL of virus diluent was added to each well, and four dilutions were set up with a dilution factor of 3. The cells were mixed crosswise and incubated in a 37 °C, 5% CO2 cell culture incubator for 48 h ± 2 h. After culture, the cells were collected, labeled with antibodies, and detected by flow cytometry. The virus titer was calculated based on the positive rate.

[0038] Data selection for titer calculation: Select groups with a positive rate between 1% and 20% for titer calculation; Titer calculation formula: Titer (TU / mL) = N*P*D / V in: N = Number of cells before lentivirus infection; P = Cell positivity rate (1%~20%) V = Volume of cells infected with lentivirus per well; D = dilution factor; for a 10-fold dilution, D = 10; for a 100-fold dilution, D = 100. TU = Transduction unit.

[0039] The flow cytometry results are shown in Table 5 below. The viral titer is 2.54E+08TU / ml.

[0040] Table 5: Flow Cytometry Results Figure 4-8 The results of flow cytometry analysis of Jurkat cells infected with the virus were obtained by adding 100 μL to dilute the cells 20, 540, 1620, and 4860 times, respectively.

[0041] (2) Detection of host DNA in the virus DNA was extracted using the Host Cell Residual DNA Sample Pretreatment Kit (Magnetic Bead Method) (Catalog No. SK030203D100, Huzhou Shenke Biotechnology Co., Ltd.). 50 μL of sample was used for extraction, followed by 50 μL of elution buffer. Other procedures were performed according to the kit instructions. Residual host DNA in the viral vector was detected using the Huzhou Shenke Biotechnology Co., Ltd. HEK293 Residual DNA Detection Kit (PCR-Fluorescent Probe Method) (Catalog No.: 1101104).

[0042] The residual value of host HEK293T DNA was detected by qPCR amplification and was 3.51E6 fg / ul.

[0043] (3) Detection of plasmid residues in the virus Plasmid remnants of the viral vector were detected using the Ori primer detection system. The amplification system and amplification standard curve were configured according to Example 1. Virus samples were serially diluted 10-fold with nuclease-free water, and plasmid remnants were detected at 10-fold, 100-fold, 1000-fold, 10000-fold, and 100000-fold dilutions. The results are shown in […]. Figure 9 .

[0044] Table 6: Results of plasmid residue detection after viral dilution from Figure 9 It is evident that amplification is inhibited at a 10-fold dilution. As the dilution factor increases, the PCR-inhibiting components in the viral vector are diluted, leading to higher detection values. However, further increases in dilution factor also result in sample loss. Based on the detection results, a 1000-fold dilution is optimal for detection. Calculations based on the dilution factor indicate a plasmid residual concentration of 2.82E7 copies / ul in the lentivirus.

[0045] 2) T cell activation and viral transduction (1) Isolation of mouse CD4+ / CD8+ cells Peripheral blood and spleens from 127 immunodeficient mice were collected using NCG. Peripheral blood cells (PBMCs) were isolated under aseptic conditions using mouse lymphocyte separation medium (Shenzhen Dakwei Biotechnology Co., Ltd., catalog number DKW33-R0100) according to the manufacturer's instructions. CD4+ / CD8+ cells were sorted using CD4 / CD8 (TIL) MicroBeads, mouse (Miltenyi, USA, catalog number 130-116-480). A total of 96 ml of CD4+ / CD8+ cells was obtained, with a cell density of 2E6 / ml.

[0046] (2) Cell activation Cell activation was performed using the C-Pro CT-60.1 Processing Kit (Cytiva, USA) on the Sepax C-Pro Cell Processing System (Cytiva, USA). Cells were resuspended at a density of 2E6 cells / ml in X-VIVO medium (containing 200 IU / mL IL-2 and 2% CTS serum substitute) for activation. 400 μL of Dynabeads™ mouse T activator CD3 / CD28 (Thermofisher, USA, catalog number 11456D) was added. After 30 min of activation in C-Pro, the activated cells were transferred to G-Rex® 100M-CS (Wilson Wolf, USA, catalog number 81100-CS). The G-Rex was then transferred to 37°C. 1 C, 5 Incubate in a 0.5% CO2 incubator for 24 hours. 6 h.

[0047] (3) Viral transduction Thaw the lentivirus prepared in step (1) at room temperature. Use a sampling bag to collect samples into a cell counter for counting. The results are as follows: Total cell density: 1.88E6 / mL viable cell density: 1.68E6 / mL Total viable cell count: 1.68E8 (96 mL) Add 1.27 ml of virus at a viral multiplicity of infection (MOI) ratio of 2:1 (2.54E8 TU / ml). Add 64 ml of X-VIVO medium (containing 200 IU / mL IL-2 and 2% CTS serum substitute) (Lonza Biotechnology, Inc., catalog number BE02-053Q) to adjust the cell density to 1E6 cells / ml. Gently mix the cells horizontally in a circular motion using a G-Rex cell culture apparatus to disperse any cells that may have clumped at the bottom. Then transfer to a 37°C, 5% CO2 incubator and incubate for 48 hours. Transduction is complete.

[0048] 3) T cell expansion culture perfusion and washing / filling Using the GatheRex Liquid Handling device (Wilson Wolf, catalog number 80000E), squeeze the liquid from the G-Rex cell culture device flask into the transduced cell storage bag. Place the cell storage tape on the magnetic plate of the magnetic rack (CTS™ DynaMag™ Magnet, catalog number: 12102, Thermo Scientific, USA) and remove the activation beads. After removing the beads, place the 10 L cell bag on the matching tray and connect the Xuri Cell Expansion System (Xuri Cell Expansion System W25, Cytiva, USA) to the waste bag (20-30 L). After locking the connectors of the culture medium and the demagnetized bead bag, connect them using a Y-connector and hang them on the rack for liquid infusion. Set the Xuri W25 parameters and introduce the cell suspension and culture medium into the Xuri cell bag using a peristaltic pump, bringing the total volume of the bag to 1 L and the cell density to approximately 1E6 / mL. After 8 days of perfusion culture, when the culture volume reaches 10 L, harvest the cells. A 3ml sample was taken from the sampling bag for flow cytometry analysis to determine the positivity rate and cell count. The viable cell density was 9.13E5 cells / ml, and the total number of viable cells was approximately 1E10 cells. The flow cytometry positivity rate was 91%.

[0049] Expanded transduced T cells were harvested using a Sefia S-2000 cell processing system (Cytiva, USA) and its accompanying FlexCell software. After concentration, washing, and freezing preparation, 350 ml of viable cells with a cell density of 2E7 cells / ml were obtained and aliquoted into seven 50 ml cell cryopreservation bags. The cell cryopreservation bags were frozen in a VIA Freeze Quad and then transferred to a liquid nitrogen storage container.

[0050] 4) Measurement values ​​of P2 and R2-TRUE of T cell preparations One bag of frozen cells was used for plasmid and host DNA residue detection. After thawing and mixing the cell preparation, 50 μL of the sample was used to extract DNA using the Host Cell Residual DNA Sample Pretreatment Kit (Magnetic Bead Method) (Huzhou Shenke Biotechnology Co., Ltd., Catalog No. SK030203D100). The sample was eluted with 50 μL of elution buffer, and other procedures were performed according to the manufacturer's instructions. After extraction, plasmid residue was detected using the Ori primer system, and host 293T DNA residue was detected using the Human Residual DNA Detection Kit (PCR-Fluorescent Probe Method) from Huzhou Shenke Biotechnology Co., Ltd.

[0051] Plasmid remnants of the viral vector were detected using the Ori primer detection system. The amplification system and amplification standard curve were prepared according to Example 1. The plasmid remnant detection value was 1.87 E3 copies / ul, and the results are shown in Table 7.

[0052] Table 7: Results of Plasmid Residue Detection The mouse TCR-T cell preparation prepared in step (3) is shown in the amplification diagram of its plasmid residues. Figure 10 .

[0053] The host DNA residue in the cell preparation prepared in step (3) was detected using the Human Residual DNA Detection Kit (PCR-Fluorescent Probe Method) from Huzhou Shenke Biotechnology Co., Ltd., and the result was 2.32E2 fg / ul.

[0054] 5) Comparison of R2 calculated by P2 / P1*R1= R2 with the actual measured R2-TRUE In step (1), 1.27 ml of viral vector was used for transduction, and in the final step (3), 350 ml of cell preparation was obtained. Residual host DNA and plasmids in the lentivirus were introduced into the cells during the viral transduction process. As the cells expanded, perfused, and were finally washed to produce the cell preparation, the residual plasmids and host DNA decreased.

[0055] The ratio of total residual plasmid in the cell preparation (P2) to total residual plasmid in the lentivirus (P1) is 1.83%. The ratio of total residual host DNA in the cell preparation (R2) to the total residual host DNA in the lentivirus (R1) is 1.82%. The calculation results show that the remaining proportions of host DNA and plasmid DNA are the same, indicating that the removal rate of DNA residue is consistent throughout the entire cell processing. The residual host DNA in the cell preparation can be calculated based on the remaining proportion of plasmid in the cell preparation and the amount of host DNA remaining in the lentivirus, i.e., R2 = R1 * (P2 / P1). The comparison results are shown in Table 8 below.

[0056] Table 8: Comparison Results of Residual Amounts Example 3: Mouse TCR-T cells were prepared manually, and the remaining proportion of residual DNA was detected and compared. 1. Isolation of mouse PBMCs 0.8 ml of peripheral blood was drawn from immunodeficient mice with OTI, and PBMCs were isolated under aseptic conditions using mouse lymphocyte separation medium (Shenzhen Dakwei Biotechnology Co., Ltd., catalog number DKW33-R0100) according to the instructions.

[0057] 2. In 24-well plates, seed 1E6 PBMC cells isolated in step 1 with 1 mL of 200 U / mL Recombinant mouse IL-2 (Thermofisher, Inc., catalog number PMC0021) and Advanced RPMI 1640 medium (Thermofisher, Inc., catalog number 12633-012). At the same time, add 2 μL of Dynabeads™ mouse T activator CD3 / CD28 (catalog number 11456D, Thermofisher, Inc.) and incubate in a 5% CO2 incubator at 37°C.

[0058] 3. After 48 hours, based on the cell count, the virus was added at an MOI of 2:1. The total cell count was 7.5E5, and 5.9 μL of the viral vector used in Example 2 (infectious titer 2.54E8 TU / mL) was added. After culturing for 24 hours in a 5% CO2 incubator at 37°C, the cells were centrifuged at 300g, the supernatant was discarded, and the cells were resuspended in 1 mL of 200 U / mL Recombinant mouse IL-2 (Thermofisher, Inc., catalog number PMC0021) and Advanced RPMI 1640 medium (Thermofisher, Inc., catalog number 12633-012). The cells were then cultured again in a 5% CO2 incubator at 37°C.

[0059] 4. Cell counts were performed every two days thereafter. When the cell proliferation reached 2.5E6 cells / mL, the cell density was maintained at 0.5E6 / mL using Advanced RPMI 1640 medium containing 200 U / mL Recombinant mouse IL-2 (Thermofisher, Inc., catalog number PMC0021). On the tenth day after virus transfection, cells were harvested, centrifuged at 300g, washed twice with PBS buffer (Thermofisher, Inc., catalog number 10010-023), counted, and resuspended in cryopreservation medium (Shenzhen Dakewei Biotechnology Co., Ltd., catalog number UH-M1002-050) to obtain 8.5 mL of finished cell product with a density of 1E7 / mL. The flow cytometry positive rate was 90.1%.

[0060] The flow cytometry results of the finished cell product are shown below. Figure 11 The positive rate was 90.1%.

[0061] 5. Detect plasmid residues of the viral vector using Kan primers, and prepare the amplification system and amplification standard curve according to Example 1. Virus sample processing: Samples were serially diluted 10-fold with nuclease-free water, and then diluted 1000-fold before detection.

[0062] Total residual plasmid in the virus = Detected concentration * Dilution factor (1000) * Viral vector volume The results of calculating the total amount of plasmid residue in the virus are shown in Table 9 below.

[0063] Table 9: Calculation results of total residual plasmids in the virus The amplification diagram of plasmid residue detection of the viral vector using Kan primers in this embodiment is shown below. Figure 12 .

[0064] Take 50 μL of cell preparation and extract DNA using the Host Cell Residual DNA Sample Pretreatment Kit (Magnetic Bead Method) (Huzhou Shenke Biotechnology Co., Ltd., Catalog No. SK030203D100). Elute with 50 μL of elution buffer, and follow the instructions for other procedures. Detect plasmid residues in the finished cell product using Kan primers, and prepare the amplification system and amplification standard curve according to Example 1. The results of the total plasmid residue detection are shown in Table 10 below.

[0065] Table 10: Results of Total Plasmid Residue Detection The amplification diagram of plasmid residues in the finished cell product detected by the Kan primer system is shown below. Figure 13 .

[0066] 6. The DNA extracted in step 5 using the Human Residual DNA Detection Kit (PCR-Fluorescent Probe Method) from the Host Cell Residual DNA Sample Pretreatment Kit (Magnetic Bead Method) (Huzhou Shenke Biotechnology Co., Ltd., Catalog No. SK030203D100) from Huzhou Shenke Biotechnology Co., Ltd. was detected to determine the residual host DNA in the final cell product. The detection value was 3.38E2 fg / ul.

[0067] The total residual plasmid DNA in the virus, P1 = 1.71E8 copies; The total residual plasmid DNA in the finished cell product was P2 = 2.43E7 copies. As shown in Example 2, the total amount of residual host DNA in the virus, R1 = 3.51E6 fg / ul * 5.9ul = 2.07E7 fg; The total amount of residual host DNA in the finished cell product, R2 = 3.38E2 fg / ul * 8.5ml = 2.87E6 fg.

[0068] The ratio of the total residual plasmid DNA P2 in the cell product to the total residual plasmid DNA P1 in the virus (P2 / P1=14.21%) was almost equal to the ratio of the total residual host DNA R2 in the cell product to the total residual host DNA R1 in the virus (R2 / R1=13.86%), with a difference of less than 5%.

[0069] The above results indicate that after viral transduction, cell amplification culture, washing, and other steps, the reduction ratio of host DNA residue and plasmid DNA residue is equal.

[0070] The difference between the host DNA clearance rate calculated based on host DNA residue and the plasmid DNA clearance rate calculated based on plasmid results is within 5%. This indicates that changes in plasmid DNA can reflect changes in host HEK293T DNA, making it feasible to calculate host DNA residue in the final cell product based on plasmid changes.

[0071] Example 4: Application of this method to detect and calculate host DNA residues in the finished product of TCR-T in actual production (practical application in cell preparation production). 1. Virus preparation status: Titer P1 R1 The titer of the lentivirus product was 5.15 × 10⁻⁶. 8 TU / mL.

[0072] 1) Detection of host DNA in the virus DNA was extracted using the Host Cell Residual DNA Sample Pretreatment Kit (Magnetic Bead Method) (Huzhou Shenke Biotechnology Co., Ltd., Catalog No. SK030203D100). 50 μL of sample was used for extraction, followed by 50 μL of elution buffer. Other procedures were performed according to the kit instructions. Residual host DNA in the viral vector was detected using the Human Residual DNA Detection Kit (PCR-Fluorescent Probe Method) from Huzhou Shenke Biotechnology Co., Ltd.

[0073] qPCR amplification revealed that the residual host HEK293T DNA was 3.67E4 fg / ul.

[0074] 1) Detection of plasmid residues in the virus Viral vector plasmid residues were detected using both the Ori and Kan primer systems. The final viral product was diluted 1000-fold and amplification was performed according to Example 1. The total viral vector count results are shown in Table 11 below.

[0075] Table 11: Results of Total Viral Vector Detection The amplification diagram of residual lentiviral plasmid detected by Ori primers is shown in the figure. Figure 14 The amplification diagram of residual lentiviral plasmids detected by Kan primers is shown in the figure. Figure 15 .

[0076] 2. Cell preparation production process 1) T cell activation CD3 cells were activated by adding 3E8 CD3-positive cells (with an error range of ±10%) to CD3 / CD28 activation beads to achieve a 1:1 ratio of CD3 cells to beads. The cells were incubated for 30 minutes in a Sepax Cpro cell culture system (Cytiva, USA) at a density of 3E6 CD3-positive cells / mL. The cells were then transferred to a G-rex cell culture apparatus, with an activation density of 2E6 cells / mL. The G-rex cell culture apparatus was then transferred to a 37°C, 5% CO2 incubator for 24 hours to complete the activation process.

[0077] 2) T cell transduction Samples were taken from G-Rex cells using a sampling bag and counted in a cell counter. The results are as follows: Total cell density: 1.63E6 / mL viable cell density: 1.16E6 / mL Total viable cell count: 1.74E8 (150 mL).

[0078] The lentiviral vector was thawed at room temperature, and then 0.68 ml of lentivirus was added according to the viral titer report at an MOI of 2. The quantitative lentiviral vector was then mixed with the transduction medium to prepare the viral vector dilution solution.

[0079] The cell-virus vector mixture was transferred to a G-rex cell culture device via a tube-connector, and the cell density was adjusted to 1E6 cells / mL using X-VIVO medium (containing 200 IU / mL IL-2 as a 2% CTS serum substitute). The G-rex cell culture device was then transferred to a 37±1°C, 5±0.5% CO2 incubator and cultured for 48±6 h to complete the transduction process.

[0080] The transduced cells were removed from the CO2 incubator, and the density was adjusted to 1E6 cells / mL with culture medium. The G-rex cell culture device was then transferred to a 37°C, 5% CO2 CO2 incubator for 24 hours.

[0081] 3) Cell expansion Using the GatheRex Liquid Handling device (Wilson Wolf, catalog number 80000E), squeeze the liquid from the G-Rex vial into the transduction cell storage bag. Place the cell storage strip on the magnetic plate of the magnetic rack (CTS™ DynaMag™ Magnet, catalog number: 12102, Thermo Scientific, USA) and remove the activation beads. After removing the beads, place the 10 L cell bag on the matching tray and connect the waste bag (20-30 L) using the Xuri Cell Expansion System (Xuri Cell Expansion System W25, Cytiva, USA). After locking the connectors of the culture medium and the demagnetized bead bag, connect them using a Y-connector and hang them on the rack for liquid inlet. Set the Xuri W25 parameters and introduce the cell suspension and culture medium into the Xuri cell bag using a peristaltic pump, bringing the total volume of the bag to 1 L and the cell density to approximately 1E6 / mL. Culture from 1000 mL to 5000 mL, and maintain the 5000 mL culture volume until the number of CAR-positive cells is ≥1.5E10 cells.

[0082] Expanded transduced T cells were harvested using a Sefia S-2000 cell processing system (Cytiva, USA) and its accompanying FlexCell software. After concentration, washing, and freezing, 352 ml of viable cells with a cell density of 5E7 cells / ml were obtained and aliquoted into seven 50 ml cell cryopreservation bags. The cell cryopreservation bags were frozen in a VIA Freeze Quad and then transferred to a liquid nitrogen storage container. Flow cytometry analysis of the cell preparation showed a positive rate of 83.1%. Results are shown below. Figure 16 .

[0083] 3. Calculated values ​​of cell preparations P2 and R2 1) Detection of plasmid residues in cell preparations The residual amount of plasmid in the cell product was detected using the Ori primer system and the KanR primer system, respectively. DNA was extracted using the host cell residual DNA sample pretreatment kit (magnetic bead method) (catalog number SK030203D100, Huzhou Shenke). 50 μL of sample was used for extraction, and 50 μL of elution buffer was used for elution. Other procedures were performed according to the kit instructions. The total amount of residual plasmid in the virus = detection concentration * dilution factor (1000) * viral vector volume. The total amount of residual plasmid in the cell product = detection concentration * dilution factor (1) * cell product volume. The detection results are shown in Table 12 below.

[0084] Table 12: Results of total plasmid residue detection in finished cell products The amplification diagram of plasmid residues in the cell product detected by the Ori primer system is shown below. Figure 17 The amplification diagram of plasmid remnants in the finished cell product detected by the Kan primer system is shown below. Figure 18 .

[0085] Based on the Ori assay results, the total residual plasmid content in the finished cell product (P2) was equal to the total residual plasmid content in the lentivirus (P1) at 1.93%. Based on the KanR gene detection results, the total residual plasmid content in the finished cell product (P2) was equal to the total residual plasmid content in the lentivirus (P1) at 1.85%. The residual host DNA concentration in the lentivirus was 3.67E4 fg / ul, with a total viral load of 680ul. The total residual host DNA R1 in the lentivirus was 2.50E7 fg.

[0086] Based on the Ori test results, and according to the formula R2=R1*(P2 / P1), the total amount of host DNA residue R2 in the cell preparation was calculated to be 4.83E5 fg.

[0087] Based on the KanR gene detection results, and according to the formula R2=R1*(P2 / P1), the total amount of host DNA residue R2 in the cell preparation was calculated to be 4.63E5 fg.

[0088] The residual host DNA in the viral vector was 25 ng, exceeding the guideline requirement of less than 10 ng / dose. The residual host DNA in the finished cell product was less than 1 ng, far below the guideline limit. Different production processes during the transformation from viral vector to finished cell product result in varying degrees of residual host DNA introduced into the viral vector. By comparing the total amount of plasmid residue in the cell preparation with the total amount of plasmid residue introduced into the lentivirus, the residual host DNA in the finished cell product can be accurately assessed.

[0089] The above description is only a partial embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0090] SEQ ID NO.1 gene sequence TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA SEQ ID NO.2 Amino acid sequence SRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF SEQ ID NO.3 KanR gene sequence 1 ATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCACTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA SEQ ID NO.4 KanR gene sequence 2 ATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCTGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA SEQ ID NO.5 KanR gene sequence 3 ATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA。

Claims

1. A method for detecting residual host DNA in cell preparations or viral preparations, characterized in that... Includes the following steps: The method involves detecting host DNA residue R1, plasmid residue P1, and plasmid residue P2 in a viral preparation, and then calculating the host DNA residue in the cell preparation using the following formula: R2 = R1 * (P2 / P1), wherein the plasmid target for detecting plasmid residue is selected from one or both of the plasmid Ori element and the KanR gene, and wherein the Ori element has the gene sequence of SEQ ID NO.1, and the KanR gene has a gene sequence capable of encoding the Kan R peptide chain.

2. The method according to claim 1, characterized in that, Residual DNA detection kits were used to detect plasmid DNA residues in viral preparations (R1).

3. The method according to claim 1 or 2, characterized in that, The KanR gene sequence is the sequence encoding SEQ ID NO.

2.

4. The method according to any one of claims 1-3, characterized in that, The KanR gene sequence has the gene sequence of SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

5.

5. The method according to any one of claims 1-4, characterized in that, The cell preparation is selected from TCR-T, CAR-T, CAR-NK and CAR-M cell preparations, and / or the viral preparation is selected from lentivirus, adenovirus and adeno-associated virus.

6. The method according to any one of claims 1-5, characterized in that, Further includes: Design primers targeting the Ori element or the KanR gene; Quantitative standards containing Ori and / or KanR targets were prepared using plasmid vectors. The primers and probes of the Ori or KanR genes were prepared into a primer mixture, added to the amplification mixture, and amplification was performed to obtain the amplification standard curve. The plasmid residue (P1) in the viral preparation was calculated based on the amplification standard curve.

7. The method according to claim 6, characterized in that, For the plasmid Ori element, the primers and probes include sequences SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8; for the plasmid KanR gene, the primers and probes include sequences SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.

11.

8. The method according to any one of claims 1-7, characterized in that, The amplification is performed by PCR, preferably qPCR.

9. The method according to any one of claims 1-7, characterized in that, The method is also used to detect plasmid DNA residues in cell preparations, plasmid DNA residues in viral preparations, and plasmid DNA residues in viral vectors.

10. A kit for detecting residual host DNA in a cell preparation or viral preparation, comprising primer probes targeting the Ori element or KanR gene, and instructions for performing the method of any one of claims 1-8 to detect residual host DNA in a cell preparation or viral preparation.

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