Immune cell carrier protective agent

Abstract

The invention discloses an immune cell carrier protective agent which is a retrovirus carrier, and belongs to the technical field of biological immunotherapy. The method involves a specific formulation of a retroviral vector centrifugation protective agent and a resuspension protective agent. The method comprises the following steps: resuscitating a PG13 toxin-producing cell strain, carrying out cell passage amplification culture, filtering a virus solution, mixing with a centrifugal protective agent, centrifuging, resuspending virus particles by using a resuspending protective agent, cryopreserving and the like, so that many problems of a traditional retrovirus vector concentration and purification method are effectively solved. Experiments show that the method can significantly improve the titer (about 3-4 times) of the retrovirus vector, greatly reduce bovine serum albumin (BSA) residue (about 99%), host DNA residue (about 65%) and host protein residue (about 99%), has important significance in the field of biological immunotherapy, and can provide powerful support for preparation of high-quality retrovirus.

Description

Technical Field

[0001] This invention relates to the field of bioimmunotherapy technology, and in particular to a protective agent that stabilizes intermediate products in the purification process of retroviral vectors. Background Technology

[0002] (I) The Importance of Retroviruses

[0003] Retroviruses play a crucial role in cell therapy. In gene therapy, they serve as highly effective vectors, their ability to infect a wide range of cell types (such as T cells and NK cells) creates favorable conditions for the precise delivery of therapeutic genes into diseased cells. Using reverse transcriptase, the viral RNA genome is transcribed into DNA and integrated into the host cell genome, allowing for the long-term stable expression of therapeutic genes and thus effectively treating diseases. At the cellular modification level, retroviruses can be used to modify immune cells, such as by introducing cytokine genes, significantly enhancing the anti-tumor activity of immune cells and providing new strategies for cancer treatment. Furthermore, the modification of stem cells is another important application area; by introducing specific genes to alter stem cell function and characteristics, they provide strong support for tissue regeneration and repair. Moreover, disease models constructed using retroviruses can accurately simulate disease processes, providing important evidence for researchers to delve into the pathogenesis of diseases and laying a solid foundation for developing more targeted and effective treatments.

[0004] (ii) Defects of unconcentrated and unpurified retroviruses

[0005] Unpurified retroviruses have several drawbacks. Their low titer significantly limits their application in experimental research and clinical treatment, making it difficult to achieve the desired biological effects. Unpurified viral solutions contain numerous impurities, such as BSA, host proteins, host DNA, culture medium components, and other irrelevant biomolecules. These impurities not only interfere with viral activity, reducing the accuracy and reliability of experimental results, but may also trigger adverse consequences such as immune responses in therapeutic applications. Furthermore, unpurified retrovirus solutions are unstable and easily affected by external environmental factors (such as changes in temperature and pH), leading to a rapid decline in viral activity.

[0006] (III) Existing Concentration and Purification Methods and Their Characteristics

[0007] Currently, methods for concentrating and purifying retroviruses are constantly evolving. Ultracentrifugation uses high-speed centrifugation to cause viral particles to settle to the bottom of the centrifuge tube, separating them from impurities. This method effectively concentrates the virus and increases titer, but it is complex and requires specialized equipment and techniques. Ultrafiltration uses an ultrafiltration membrane to filter the viral solution, retaining viral particles and removing small molecule impurities. This method is simple to operate but may affect viral activity. Precipitation involves adding a precipitant to precipitate viral particles, followed by separation of the precipitate. This method is relatively low-cost, but the purification effect is relatively poor. In practical applications, the appropriate purification method must be selected based on the specific circumstances. For example, ultracentrifugation and ultrafiltration are commonly used in laboratory research, while clinical treatment, due to higher requirements for viral safety and efficacy, requires more stringent purification standards.

[0008] (IV) Advantages and Precautions of Centrifugation

[0009] Centrifugation, a classic method for concentrating and purifying retroviruses, offers several advantages. It effectively increases viral titer, removes most impurities, and results in a purer virus. Centrifugation is relatively simple to perform, and different degrees of concentration and purification can be achieved by adjusting centrifugation parameters. Furthermore, centrifugation equipment is readily available and relatively inexpensive, making it suitable for widespread application. When using centrifugation, attention must be paid to the centrifugation speed and time, the centrifugation protectant, and the resuspending agent. Appropriate centrifugation speed and time ensure sufficient sedimentation of viral particles without affecting their activity. A suitable centrifugation protectant maintains viral stability, while optimizing the resuspending agent ensures good viral dispersion. In addition, combining viral titer determination and impurity analysis with other detection methods allows for a comprehensive evaluation and optimization of the purification effect, leading to high-quality retroviruses.

[0010] There are currently no reports on retrovirus purification methods as described in this application. Summary of the Invention

[0011] This invention provides a retrovirus concentration and purification protectant, which is particularly suitable for the concentration and purification of retroviruses.

[0012] In one or more embodiments, the virus concentration and purification method employs a specific formulation of a centrifugation protectant comprising DMEM medium and a 5%-15% sucrose solution. Experimental screening has determined that this formulation significantly improves the stability of the virus during centrifugation, thereby enhancing virus concentration and purification efficiency.

[0013] In one or more embodiments, the virus concentration and purification method uses a resuspension protectant with a specific formulation comprising DPBS buffer, 5%-15% sucrose solution, and 1mM-150mM NaCl. Through a series of experimental screenings, this formulation has been shown to effectively improve the stability of the virus after centrifugation, ensuring the effectiveness of virus concentration and purification.

[0014] In one or more embodiments, the virus concentration and purification method utilizes optimized centrifugation conditions, specifically centrifugation at 4°C and 10,000g for 4-16 hours (preferably 4 hours). These conditions ensure sufficient sedimentation of virus particles while avoiding adverse effects on viral activity due to excessively high speed and prolonged centrifugation.

[0015] In one or more embodiments, the virus concentration and purification process includes the following steps:

[0016] (1) Cell resuscitation and culture: Preheat D10 complete medium to 37°C in a water bath and add 5ml to a 15ml centrifuge tube for later use. Take out the frozen toxin-producing cell line (such as PG13 toxin-producing cells) from a -80°C freezer or liquid nitrogen, and quickly immerse it in a 37°C water bath, shaking occasionally until the cells are evenly thawed and the ice particles in the cryovial melt. After taking it out, sterilize it and transfer it to a biosafety cabinet. Unscrew the cap and add the rewarmed medium drop by drop. Gently aspirate the cell suspension, centrifuge at 450g for 5 minutes, discard the supernatant, add D10 complete medium to resuspend the cells, stain with trypan blue and count them. Take an appropriate amount of cell suspension and seed it into a T25 culture flask, add D10 complete medium and cell suspension, and incubate at 37°C in a 5% CO2 incubator. Clean up the room after the operation.

[0017] (2) Cell passage and expansion culture: Observe cell morphology and confluence in T25 culture flasks the day after resuscitation, and perform operations such as digestion, termination of digestion, centrifugation, and cell counting. If the cell number is less than 3 × 10⁻⁶ cells / year... 6 Continue seeding in T25 culture flasks; if the cell count is 3×10⁻⁶, continue seeding. 6 -6×10 6 During this period, cells can be seeded in T75 culture flasks (total volume 20ml / flask). Observe the cells in the T75 culture flasks. When the confluence reaches about 90%, stop the expansion culture and proceed to the virus collection and purification process.

[0018] (3) Virus concentration, purification, and cryopreservation: Take the virus solution from the T75 culture flask with a confluence of 90% and transfer it to a 0.45μm filter using a pipette. Mix the filtered virus solution and virus centrifugation protectant at a ratio of 3:1, and centrifuge at 10000g for 4-16 hours at 4°C. After centrifugation, discard the supernatant, add one-quarter volume of virus resuspending protectant to resuspend the virus particles, and finally transfer to a -80°C freezer for cryopreservation.

[0019] The present invention also provides a centrifugation protectant for virus concentration and purification, having a formulation as described above, namely comprising DMEM medium and 5%-15% sucrose solution.

[0020] The present invention also provides a resuspension protectant for virus concentration and purification, having the formulation described above, namely comprising DPBS buffer, 5%-15% sucrose solution and 1mM-150mM NaCl.

[0021] The advantages of this invention are as follows: Traditional retrovirus concentration and purification methods have many limitations, such as the cumbersome operation and stringent equipment requirements of ultracentrifugation, the potential impact of ultrafiltration on viral activity, and the unsatisfactory purification effect of precipitation. The method of this invention, through careful optimization of the centrifugation protectant, resuspension protectant formulation, and centrifugation conditions, significantly improves viral titer (approximately 3-4 times higher after concentration and purification) while effectively reducing residual impurities (e.g., approximately 99% reduction in BSA residue, approximately 65% ​​reduction in host DNA residue, and approximately 99% reduction in host protein residue). Furthermore, the method of this invention is relatively simple to operate, low in cost, and requires less sophisticated equipment, making it more suitable for widespread application. Cell killing and targeted proliferation experiments have demonstrated that the chimeric antigen receptor T cells (CAR-T cells) prepared based on the method of this invention function well, contributing to their better role in the field of biological immunotherapy and providing a more effective means of treating related diseases, thus meeting the urgent need for high-quality retroviruses. Attached Figure Description

[0022] Figure 1 Description diagram of the invention point

[0023] Figure 2 The positive rate of CAR expression in each group of retrovirus centrifugation protectants;

[0024] Figure 3 The positive rate of CAR expression in each group of retrovirus resuspending protectants;

[0025] Figure 4 Comparison of virus titer detection data before and after virus concentration and purification;

[0026] Figure 5 Comparison of BSA residue detection data before and after virus concentration and purification;

[0027] Figure 6 Comparison of viral host DNA detection data before and after viral concentration and purification;

[0028] Figure 7 Comparison of viral host protein detection data before and after viral concentration and purification;

[0029] Figure 8 The results of cell killing experiments before and after virus concentration and purification;

[0030] Figure 9 The results of CAR+ cell-targeted proliferation experiments before and after virus concentration and purification are shown. Detailed Implementation

[0031] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0032] Example 1: Screening test of virus centrifugation protectant formulation

[0033] 1. Experimental Methods

[0034] First, the frozen PG13 virus-producing cell line was removed from its cryogenic environment and quickly thawed in a 37°C water bath. After the cells were uniformly thawed, they were inoculated into T25 culture flasks pre-filled with an appropriate amount of preheated D10 complete medium. Next, the culture flasks were placed in an incubator at 37°C and 5% CO2 for culture. During this period, the cell growth status was closely monitored, and passage was performed as needed to ensure continuous cell proliferation. When the cell confluence reached approximately 90%, the virus solution was collected, sterilized using a 0.45 μm filter membrane, and then concentrated by centrifugation using a virus centrifugation protectant. After centrifugation, the virus was resuspended and stored using a virus resuspending protectant (as shown in Table 1).

[0035] During the virus centrifugation and concentration process, 16 virus centrifugation protectant formulations were designed using the Design of Experiments (DOE) method (e.g., ...). Figure 2 As shown in the image, these formulations all used DMEM as the base buffer, with different concentrations of sucrose solution, Tris-HCl solution, NaCl solution, MgCl2 solution, and EDTA solution added. The filtered virus solution was carefully mixed with 16 different centrifugation protectants at a 3:1 volume ratio on ice. The mixture was then transferred to high-speed refrigerated centrifuge tubes and centrifuged at 4°C and 10,000g for 4 hours. After centrifugation, the supernatant was discarded, and the precipitate was gently resuspended in DPBS buffer to obtain the virus solutions with different treatments. Finally, these virus solutions were used to infect activated human T cells in preparation for subsequent assays.

[0036] Table 1 Grouping of Screening Tests for Retroviral Centrifugation Protectant Formulations

[0037] Group sucrose(%) Tris-HCl (mM) <![CDATA[ Nacl (mM)]]> MgCl2 (mM) EDTA (mM) 1 5 0 0 0 1 2 5 0 0 2 0 3 5 0 100 0 0 4 5 0 100 2 1 5 5 30 0 0 0 6 5 30 0 2 1 7 5 30 100 0 1 8 5 30 100 2 0 9 15 0 0 0 0 10 15 0 0 2 1 11 15 0 100 0 1 12 15 0 100 2 0 13 15 30 0 0 1 14 15 30 0 2 0 15 15 30 100 0 0 16 15 30 100 2 1

[0038] 2. Experimental Results

[0039] Two days after retroviral infection of activated human T cells, flow cytometry was used to analyze the cells to determine CAR. + Cell proportion. NT values ​​of 1×10⁻⁶ cells were collected from the chimeric antigen receptor group targeting the CD19 CAR structure and the negative, uninfected control group.6 Cells were centrifuged to remove the culture medium, washed once with 500 μl PBS, and resuspended in 100 μl of PBS in a flow cytometry tube (BD). Primary antibody Anti-Mouse FMC63scFv Polyclonal Antibody (1:100) was added and incubated at °C for 30 min. Cells were then washed with 500 μl PBS, followed by BV421 Streptavidin (1:100), washed again with 500 μl PBS, and resuspended in 200 μl PBS for flow cytometry analysis. The results showed that CAR cells in group 9... + The proportion is relatively high (e.g.) Figure 2 As shown in the figure, and with relatively simple composition, DMEM medium containing a final concentration of 5-15% sucrose solution was finally determined as the centrifugation protectant for retroviruses.

[0040] Example 2: Screening test of retroviral virus resuspension protectant formulation

[0041] 1. Experimental Methods

[0042] The viral suspension was treated using the retroviral centrifugation protectant formulation screened in Example 1, and viral precipitate was obtained after centrifugation. Next, using the Design of Epithet (DOE) method again, 21 viral resuspending protectant formulations were designed (as shown in Table 2). These formulations all used DPBS as the base buffer and added different concentrations of sucrose solution, Tris-HCl solution, NaCl solution, MgCl2 solution, and EDTA solution. The viral precipitate was resuspended using these 21 different resuspending protectants. The resuspended viral suspension was transferred to sterile cryovials, labeled, and stored at -80°C. After cryopreservation, the viral suspension was removed from the freezer and used to infect activated human T cells.

[0043] Table 2 Grouping of Retroviral Resuspension Protectant Formulation Screening Tests

[0044] Group sucrose(%) Tris-HCl (mM) <![CDATA[ Nacl (mM)]]> MgCl2 (mM) EDTA (mM) 1 5 0 0 0 0 2 5 0 0 2 1 3 5 0 150 0 1 4 5 0 150 0 1 5 5 0 150 2 0 6 5 20 0 0 0 7 5 20 0 2 0 8 5 20 0 2 1 9 5 20 150 0 1 10 5 20 150 2 0 11 15 0 0 0 0 12 15 0 0 2 0 13 15 0 0 2 1 14 15 0 150 0 0 15 15 0 150 0 0 16 15 0 150 2 1 17 15 20 0 0 1 18 15 20 0 0 1 19 15 20 150 0 0 20 15 20 150 2 0 21 15 20 150 2 1

[0045] 2. Experimental Results

[0046] Two days after retroviral infection of activated human T cells, flow cytometry was used to analyze the cells to determine CAR. + Cell proportion. NT values ​​of 1×10⁻⁶ cells were collected from the chimeric antigen receptor group targeting the CD19CAR structure and the negative, uninfected control group. 6Cells were centrifuged to remove the culture medium, washed once with 500 μl PBS, and resuspended in 100 μl of PBS in a flow cytometry tube (BD). Primary antibody Anti-Mouse FMC63scFv Polyclonal Antibody (1:100) was added and incubated at °C for 30 minutes. Cells were washed with 500 μl PBS, then BV421 Streptavidin (1:100) was added, followed by washing with 500 μl PBS. Cells were then resuspended in 200 μl PBS and analyzed. (e.g.) Figure 3 (As shown) Experimental results show that CAR in groups 14 and 15 + The proportion was significantly higher than in other groups. Therefore, a DPBS solution containing 5%-15% sucrose solution and 1mM-150mM NaCl solution was selected as the formulation for retrovirus resuspending protection.

[0047] Example 3: Verification Experiment of Retrovirus Purification Efficiency

[0048] 1. Virus titer detection

[0049] Accurately measure the virus solution before and after concentration and purification, and perform 10-fold serial dilutions using complete culture medium, starting from 10... -1 Up to 10 -6 Six dilutions were set up, with three replicates for each dilution. The diluted virus solution was added to each 24-well plate, with 100 μl of virus solution and 900 μl of 293T cell suspension added to each well (1*10^5 / well). Polybrene was added to each well to a final concentration of 5 μg / ml. A blank control well containing only culture medium was also included. The plates were centrifuged at 32°C, 2500 RPM for 90 min. Two days later, flow cytometry was used to detect CAR on the surface of 293T cells. + For specific detection methods, please refer to CAR in Example 1. + The detection. By calculating CAR + The viral titer was calculated by combining the proportion of cells, viral dilution, and the number of infected 293T cells (viral titer = viral volume × CAR). + (ratio / number of infected cells). Experimental results showed that the retrovirus titer increased by approximately 3-4 times after concentration and purification (e.g., ratio / number of infected cells). Figure 4 As shown in the figure, the concentration was significantly higher than before concentration and purification. This result fully demonstrates that the concentration and purification method of the present invention has a significant effect on increasing virus concentration, enabling the virus to maintain a high infectivity at a lower volume, thereby better meeting various experimental and therapeutic needs.

[0050] 2. BSA Impurity Residue Detection

[0051] A dedicated BSA ELISA kit was used to detect the virus solution before and after concentration and purification. First, following the kit instructions, the standards were precisely serially diluted using the diluent provided in the kit to establish a standard curve. Next, the virus solutions before and after concentration and purification were appropriately diluted with the diluent. Then, 100 μl of the diluted standards and samples were added to each well of an ELISA plate, with three replicates per sample and a blank control well. The plate was sealed with a sealing film and incubated at 37°C for 1 hour. After incubation, the sealing film was carefully removed, the liquid in the wells was discarded, and the plate was washed three times with washing buffer, soaking for 1-2 minutes each time, and then patted dry. 100 μl of enzyme-labeled secondary antibody working solution was added to each well, the plate was resealed, and incubated at 37°C for 30 minutes. After repeating the washing steps, 100 μl of substrate solution was added to each well, and the plate was incubated at 37°C in the dark for 15 minutes. Finally, add 100 μl of stop solution to each well, mix gently, and immediately measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate the residual BSA in the viral solution based on the standard curve. Experimental results show that the method of this invention can significantly reduce BSA residue by approximately 99% (e.g., Figure 5 (As shown). Low BSA residue can significantly reduce the risk of immunogenicity, effectively improve the purity and stability of viral products, and help ensure the purity and stability of viral products, reducing potential interference and adverse effects caused by BSA residue in subsequent experimental or therapeutic applications.

[0052] 3. Detection of residual host DNA impurities

[0053] DNA was extracted from the viral solution before and after concentration and purification using a specialized DNA extraction kit. Specific primers and probes were designed based on the host cell genome sequence to prepare a real-time PCR reaction system, including SYBR PremixEx Taq II (12.5 μl), forward and reverse primers (1 μl each, 10 μM), probe (1 μl, 5 μM), and template DNA (2 μl), with nuclease-free water added to a final volume of 25 μl. The reaction system was added to a dedicated reaction tube for a real-time PCR instrument, and the reaction program was set, along with a known concentration of host cell genomic DNA standard. A standard curve was established, and the residual amount of host DNA in the viral solution was calculated based on the standard curve. Experimental results showed that the method of this invention can significantly reduce the residual host DNA by approximately 65% ​​(e.g., ...). Figure 6 (As shown). Low host DNA residue helps reduce the risk of immunogenicity, ensures the purity of viral products, reduces adverse consequences such as immune responses that may be caused by host DNA residue, and improves the safety and efficacy of viral products in biological immunotherapy.

[0054] 4. Detection of residual host protein impurities

[0055] ELISA was used to detect residual host proteins. First, the virus solution before and after concentration and purification was appropriately diluted with coating buffer and then added to an ELISA plate, 100 μl per well. The plate was incubated overnight at 4°C to allow the host proteins in the virus solution to adsorb onto the plate. The next day, the liquid in the wells was discarded, and the plate was washed three times with washing buffer (e.g., PBST containing 0.05% Tween-20 phosphate buffer), soaking for 3 minutes each time, and then blotted dry. Next, 200 μl of blocking buffer (PBST containing 1% BSA) was added to each well, and the plate was incubated at 37°C for 1 hour to block non-specific binding sites. After incubation, the blocking buffer was discarded, and the plate was washed three times with washing buffer. Specific anti-host protein antibodies (primary antibody) were diluted with blocking buffer, 100 μl per well, and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with washing buffer. Then, the HRP-labeled secondary antibody was diluted with blocking buffer (the dilution ratio was determined according to the antibody instructions), 100 μl was added to each well, and incubated at 37°C for 1 hour. The plate was washed three times with washing buffer. Finally, 100 μl of substrate solution (TMB substrate solution) was added to each well, and the plate was incubated at 37°C in the dark for 15 minutes. After color development, 50 μl of stop solution was added to each well, and the mixture was gently mixed. The absorbance (OD value) was immediately measured at 450 nm using a microplate reader. The residual amount of host protein in the viral solution was calculated by comparing it with a standard curve prepared with a known concentration of host protein standard. The experimental results showed that the residual amount of host protein in the viral solution was relatively high before concentration and purification, and the residual amount of host protein in the viral solution was significantly reduced after concentration and purification, by about 99% (e.g., Figure 7 (As shown). The significant reduction in host protein residue is of great significance for improving the purity and stability of viral products, while also reducing the risk of immunogenicity, which is conducive to the virus functioning better in vivo and reduces problems such as immune rejection that may be caused by host protein residue. This further enhances the application value of the viral concentration and purification method of the present invention in the field of biological immunotherapy.

[0056] 5. Cell killing experiment

[0057] Activated T cells were infected with both pre- and post-concentrated purified retroviruses and cultured in X-VIVO15 complete medium containing 500 U / ml recombinant human IL-2 for 7-10 days to obtain CD19CAR-T cells. During culture, the medium was changed every 2-3 days, and an appropriate amount of IL-2 was supplemented to maintain good cell growth. After harvesting, cells were cultured at different effector-to-target ratios (1×10⁻⁶). 4Using CAR-T cells as a baseline, with effector-to-target ratios of 3:1, 1:1, and 1:6, CAR-T cells before and after concentration and purification, and target cells (CD19-positive target cells Nalm6-LUC-GFP) were added to 96-well plates, with multiple replicate wells for each effector-to-target ratio, and positive control wells containing only target cells. The 96-well plates were incubated overnight at 37°C in a 5% CO2 incubator. After incubation, 100 μl of luciferase reaction substrate was added to the culture system, gently mixed, and the fluorescence value was detected using a microplate reader. Based on the detected fluorescence value, the killing efficiency of CAR-T cells against target cells was calculated using the formula: Killing efficiency = [(fluorescence value of positive control well - fluorescence value of experimental well) / fluorescence value of positive control well] × 100%. The experimental results showed that the killing efficiency of concentrated and purified CAR-T cells against target cells at different effector-to-target ratios was comparable to that before purification (e.g., ...). Figure 8 As shown in the figure, this indicates that the concentration and purification method of the present invention can maintain the activity of the virus and ensure the killing function of CAR-T cells while improving the purity of the virus.

[0058] 6. Targeted proliferation capacity detection experiment

[0059] In this assay, the proliferation of CAR-T cells prepared before and after retrovirus concentration and purification, co-cultured with target cells, was observed to understand whether concentration and purification might affect viral function. Counting and flow cytometry analysis were used to provide data support for optimizing CAR-T cell therapy. In this experiment, CAR-T cells prepared before and after retrovirus concentration and purification were mixed with target cells (Nalm6) at a 1:5 effector-to-target ratio and placed in 24-well plates, incubated at 37°C in a 5% CO2 incubator. Samples were taken every 1-2 days for counting, and a portion of the cells was analyzed by antibody flow cytometry. The remaining cells were supplemented with target cells for continued culture. The fold increase of CAR-T cells was calculated based on the flow cytometry and cell counting results. The experimental results showed that the targeted proliferation capacity of concentrated and purified CAR-T cells was comparable to that before purification (e.g., ...). Figure 9 As shown in the figure, this indicates that the concentration and purification method of the present invention can maintain the activity of the virus and ensure the proliferation capacity of CAR-T cells while improving the purity of the virus.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A protective agent for the purification of retroviruses, characterized in that, The protective agents include retrovirus centrifugation protective agents and retrovirus resuspension protective agents.

2. The retrovirus centrifugation protectant according to claim 1 includes a culture medium, wherein the culture medium contains a sucrose solution with a final concentration of 5-15%.

3. The retrovirus resuspension protectant according to claim 1 comprises a buffer solution, wherein the buffer solution contains a sucrose solution with a final concentration of 5-15% and a NaCl solution with a final concentration of 1-150 mM.

4. The retroviral centrifugation protectant according to claim 2, characterized in that, The culture medium is serum-free DMEM.

5. The retroviral resuspension protectant according to claim 3, characterized in that, The buffer solution is DPBS buffer.

6. A method for preparing the retroviral centrifugation protectant as described in claim 2, characterized in that, Includes the following steps: (1) Measure out the culture medium and sucrose solution respectively, mix them evenly, and prepare a culture medium solution containing sucrose solution, which is the reverse virus centrifugation protectant.

7. A method for preparing the retroviral resuspension protectant as described in claim 3, characterized in that, Includes the following steps: (1) Measure out the buffer solution, sucrose solution and NaCl solution respectively, mix them evenly, and prepare a solution containing sucrose solution and NaCl solution; (2) Add pH adjuster to adjust the pH of the culture medium solution to 6.8-7.4 to obtain a protective agent for resuspension of retrovirus.

8. A method for purifying retroviruses, characterized in that, The process includes the following steps: premixing the retrovirus stock solution with the retrovirus centrifugation protectant described in claim 2 at a volume ratio of 1 to 3:1 to obtain a retrovirus premix solution; (1) The retrovirus stock solution and the retrovirus centrifugation protectant described in claim 2 are premixed at a volume ratio of 1 to 3:1 to obtain a retrovirus premix solution; (2) Place the retrovirus premix in a high-speed refrigerated centrifuge at 4°C and 10,000g for 4 to 16 hours; (3) Remove the retrovirus supernatant and add the retrovirus resuspending protectant described in claim 3 to the supernatant at a ratio of 1 to 3:1; (4) The retrovirus solution was filtered using a 0.45 μm filter membrane to obtain a purified retrovirus concentrate.

9. The method for purifying retroviruses according to claim 8, characterized in that, The retrovirus stock solution is obtained through the following process steps: (1) Preheat D10 medium (DMEM medium containing 10% fetal bovine serum), thaw frozen PG13 virus packaging cell line, add medium, centrifuge and resuspend, count and inoculate T25 culture flasks for culture; (2) Observe T25 cells the next day. After digestion and counting, if the number reaches the target, expand the inoculation to T75 culture flasks and observe the growth of T75 cells. When the confluence reaches about 90%, stop the expansion culture operation. (3) After terminating the expansion culture, harvest the retrovirus according to the procedure and filter it with a 0.22um filter to remove bacteria, and harvest the retrovirus stock solution.

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