Method for preparing low host DNA in a human adenovirus preparation

Abstract

The application discloses a preparation method of low host DNA in human adenovirus preparation and relates to the technical field of gene therapy preparation. The preparation method is as follows: a pretreatment agent is added into an adenovirus-containing solution for pretreatment, and then microjet treatment is performed; per liter of the adenovirus-containing solution comprises the following concentrations of the pretreatment agent: 18-30 g / L of sodium chloride, 2-5 g / L of arginine hydrochloride, 0.1-1 mL / L of non-ionic surfactant and 5-10 g / L of potassium chloride, and a PBS buffer is used to stabilize pH; the microjet treatment adopts a Y-shaped channel structure, the channel aperture is 25-100 mu m, and the pressure of the microjet treatment is 2000-3000 bar. The combination of the pretreatment and the microjet treatment achieves the technical effects of efficient removal of host DNA and efficient recovery of viruses.

Description

Technical Field

[0001] This invention relates to the field of gene therapy formulation technology, specifically a method for preparing low-host DNA in a human adenovirus formulation. Background Technology

[0002] Recombinant oncolytic adenoviruses originate from natural adenoviruses (a common type of DNA virus), which naturally possess the ability to infect and replicate within cells. Through gene editing techniques (such as deleting or modifying key genes like E1B and E3), the virus can be engineered to replicate only in tumor cells, losing its ability to replicate in normal cells. This modification leverages the characteristics of tumor cells (such as p53 dysfunction or Rb pathway abnormalities), making it a tool for targeted therapy.

[0003] In tumor cells, viruses can replicate efficiently and eventually lyse the cells, releasing new viral particles. These particles can then infect surrounding tumor cells, creating a chain reaction. This process not only directly destroys tumor tissue but also releases large amounts of tumor titers and viral components, laying the foundation for immune activation. The tumor titers and viral components (such as viral DNA or proteins) released during the lysis process strongly activate the body's immune system, particularly dendritic cells and T cells. This triggers an anti-tumor immune response (such as enhanced titer presentation and T cell killing), which, combined with direct oncolysis, forms a synergistic anti-tumor mechanism of "oncolysis + immunity," effectively eliminating residual or metastatic tumor cells.

[0004] As an important research direction in the field of tumor immunotherapy, recombinant oncolytic adenovirus represents a shift from traditional chemotherapy to targeted immunotherapy. It combines the advantages of direct cell killing and systemic immune activation, and is currently being used in clinical trials to treat various solid tumors (such as bladder cancer and melanoma), and is considered a key strategy for improving cancer treatment efficacy and reducing side effects.

[0005] In the production of adenovirus preparations, the harvested material infected with the virus contains the target adenovirus, as well as host cell debris, proteins, host DNA, and cell culture medium components. All these impurities need to be removed. Among these, the key to the safety of preparing human adenovirus preparations using 293 cells is the content of residual host DNA in the cells. The residual host DNA content in human adenovirus preparations must not exceed 10 ng / dose (PCR method), and the size of residual DNA fragments should be controlled below 200 bp.

[0006] In addressing various indications, particularly when using systemic administration, high-dose viral loads or multiple consecutive doses are often required. Against this backdrop, developing a host DNA purification process for human adenovirus formulations is crucial. This process must effectively remove residual host DNA from adenovirus formulations to meet national pharmacopoeia standards while significantly improving virus recovery rates, which is of great importance. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing low-host DNA in human adenovirus preparations, so as to solve the problems mentioned in the background art.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] A method for preparing low-host DNA in a human adenovirus preparation: by adding a pretreatment agent to an adenovirus-containing solution for pretreatment, followed by microfluidic treatment, purification, and sterilization, a low-host DNA adenovirus is obtained; subsequently, after formulation and packaging, a low-host DNA adenovirus preparation is obtained.

[0010] Furthermore, the pretreatment agent is sodium chloride; the concentration of sodium chloride in each liter of adenovirus-containing solution is 18~30 g / L.

[0011] Furthermore, the concentration of sodium chloride in each liter of adenovirus-containing solution is 24-30 g / L.

[0012] Furthermore, the preprocessing is an oscillation process, with a processing time of 2-4 hours.

[0013] Furthermore, the microjet processing adopts a Y-shaped channel structure with a channel aperture of 25~100μm, a microjet processing pressure of 2000~3000bar, and a microjet processing frequency of 1~3 times.

[0014] Furthermore, the channel aperture of the Y-shaped channel structure is 25~50μm, and the pressure of the microjets is 2500~3000bar.

[0015] Furthermore, the adenovirus-containing solution is a virus harvest solution, an ultrafiltration concentrate, or a stock solution.

[0016] Furthermore, the pretreatment agent also includes arginine hydrochloride, a nonionic surfactant, PBS buffer, and potassium chloride.

[0017] Furthermore, in each liter of adenovirus-containing solution, the concentration of arginine hydrochloride is 2-5 g / L, the concentration of nonionic surfactant is 0.1-1 mL / L, and the concentration of potassium chloride is 5-10 g / L; wherein, PBS buffer is added simultaneously with arginine hydrochloride to stabilize the pH of the adenovirus-containing solution and avoid large pH fluctuations.

[0018] Furthermore, in each liter of adenovirus-containing solution, the concentration of arginine hydrochloride is 3-4 g / L, the concentration of nonionic surfactant is 0.4-0.6 mL / L, and the concentration of potassium chloride is 7-8 g / L; wherein, PBS buffer is added simultaneously with arginine hydrochloride to stabilize the pH of the adenovirus-containing solution and avoid large pH fluctuations.

[0019] Furthermore, the nonionic surfactant is Tween 20.

[0020] Furthermore, the ultrafiltration concentrate is obtained by ultrafiltration concentration of the virus harvest fluid; the specific method is as follows: first, the virus harvest fluid is filtered using a 0.8 μm filter cartridge, and then filtered using a 0.65 μm filter cartridge to obtain a clarified virus harvest fluid; then, the clarified virus harvest fluid is ultrafiltered and concentrated 10-15 times using a 300KD low protein adsorption ultrafiltration membrane to obtain a primary concentrate; finally, the primary concentrate is washed with 6-8 volumes of 0.01 mol / L PBS buffer and then washed with 8-10 volumes of 0.35 mol / L sodium chloride solution to obtain the ultrafiltration concentrate.

[0021] Furthermore, the stock solution is obtained by purifying the ultrafiltration concentrate using molecular sieve gel chromatography. The specific method is as follows: the ultrafiltration concentrate is separated and purified by molecular sieve gel chromatography at a linear flow rate of 35~45 cm / h, and eluted with 0.01 mol / L PBS buffer while controlling the pressure to <0.8 bar. The first peak is collected to obtain the stock solution.

[0022] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0023] (1) DNA and viruses are bound together through various means such as electrostatic interaction, hydrogen bonding, and hydrophobic interaction. Therefore, sodium chloride is used as the main raw material for pretreatment in this invention. The addition of sodium chloride changes the salt ion concentration in the solution, thereby interfering with these interactions and effectively separating DNA from viruses, laying the foundation for subsequent microfluidic treatment and purification steps.

[0024] (2) Arginine hydrochloride was added in this invention. The guanidin group contained therein can work with sodium chloride to interfere with the interaction between DNA and virus, making DNA easier to detach. At the same time, arginine can form hydrogen bonds with viral capsid proteins, which helps maintain the stability of adenovirus under high salt conditions. Considering that the addition of arginine hydrochloride will affect the pH of the adenovirus-containing solution, PBS buffer needs to be added at the same time to stabilize the solution pH and avoid large pH fluctuations that may affect adenovirus activity.

[0025] (3) In this invention, a nonionic surfactant, Tween 20, is also added. Nonionic surfactants can avoid electrostatic binding with DNA and directly coat the virus surface to form a physical barrier. This further synergizes with arginine hydrochloride to maintain the stability of adenovirus and can also improve the virus recovery rate. Furthermore, nonionic surfactants can further reduce the interfacial tension of the system, allowing DNA with strong adsorption on the virus surface to be further separated, and can also prevent the virus from adsorbing residual DNA.

[0026] (4) In this invention, potassium chloride and sodium chloride are further added in synergy, because K + Na + Since their ionic radii are similar, potassium chloride can superimpose the ionic competitiveness of sodium chloride, thereby enhancing its ability to interfere with the interaction between DNA and viruses.

[0027] (5) In this invention, microfluidic treatment is further used to induce the linear DNA molecules of the host cell to break, thereby achieving the technical effect of efficient removal of host DNA and efficient recovery of virus.

[0028] (6) This invention achieves efficient removal of host DNA and efficient recovery of viruses through the synergistic effect between the components in the pretreatment agent and the control of process parameters in microfluidic treatment. Studies have found that microfluidic treatment at a pressure of 2500~3000 bar can achieve a host DNA removal rate of up to 90~99%, and the residual host DNA in each dose of product can be ≤1ng / dose, which is far below the standard stipulated by the National Pharmacopoeia. Moreover, the residual fragments are all ≤200bp, and the product quality has high stability and high controllability.

[0029] In summary, this invention achieves highly efficient removal of host DNA and high virus recovery by using sodium chloride, potassium chloride, arginine hydrochloride, and nonionic surfactants as pretreatment agents to treat virus harvest liquid, ultrafiltration concentrate, or stock solution, followed by microfluidic treatment. This is of great significance. Attached Figure Description

[0030] Figure 1 This is a process diagram for preparing low-host-DNA adenovirus preparations.

[0031] Figure 2 The results of host DNA removal rate of virus harvest fluid under different microjet pressures are shown in the figure.

[0032] Figure 3 The graph shows the host DNA removal rate of the ultrafiltration concentrate under different microjet pressures.

[0033] Figure 4 The graph shows the host DNA removal rate of the original solution under different microjet pressures. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] It should be noted that the following quantities are by weight, and there are no special restrictions on the suppliers of all raw materials involved in this invention. Exemplary examples include:

[0036] In the following embodiments, the domestically produced microfluidic device is branded as Antos and model as AMH-NANO.

[0037] Imported microfluidic equipment: brand is HS from the UK, model is SPCH-EP-IC-10-60; raw materials are all commercially available.

[0038] A method for preparing low-host DNA from a human adenovirus preparation, the specific process of which is as follows (see flowchart). Figure 1 :

[0039] I. Processing of virus harvested fluid:

[0040] Examples 1-12: A method for preparing low-host DNA in a human adenovirus preparation:

[0041] 1. Obtaining the virus harvesting solution: (1) Take a certain number of 293.2sus cells from the liquid nitrogen tank and immediately place them in a 37°C constant temperature water bath to thaw rapidly; then mix the thawed cell suspension and transfer it to a DMEM complete culture medium container, centrifuge, and then use 3~5×10 5 Seeds were seeded in shake flasks at a density of 37°C and 120 rpm, and cultured until the cell count reached 3-5 × 10⁶ cells / mL. 6 Pass the cells at a density of 3-5 × 10⁶ cells / mL. 5 Cells / mL; continue culturing at 37℃ and 120 rpm until cells grow to 3~5×10⁻⁶. 6When the cell count / mL and viability >95%, the cells were passaged into a bioreactor and cultured at 37°C, pH 7.4, and dissolved oxygen 50% until the cells grew to 3-4 × 10⁶ cells / mL. 6 When the cell / mL and the survival rate is >90%, adenovirus is inoculated at MOI=5 and cultured at 37℃, pH=7.4 and dissolved oxygen 50%; (2) After adenovirus inoculation and culture for 72h, the virus cell material is centrifuged at 4℃ and 1000rpm for 5min and virus-carrying cells and supernatant are collected; (3) The virus-carrying cells are subjected to repeated freeze-thaw cycles at 37℃ and -80℃ three times to lyse the cells and release the virus. Then, the freeze-thaw product is centrifuged at 4℃ and 2000rpm for 5min and virus is collected; (4) The virus and supernatant are mixed evenly to obtain virus harvesting liquid.

[0042] 2. The above-mentioned virus harvested fluid was divided into 3 batches, with 12 samples taken from each batch, and uniformly numbered as Example 1, Example 2, Example 3... Example 12. Different concentrations of sodium chloride were added to each batch of virus harvested fluid from Examples 1 to 5, and then microfluidic treatment was performed on them using a domestically produced microfluidic device to verify the role of sodium chloride in the virus harvested fluid and to determine the optimal concentration of sodium chloride to be added, as detailed below:

[0043] Example 1: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, microfluidic treatment was performed using a domestically produced microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and 2 treatments.

[0044] Example 2: Sodium chloride was added to the virus harvest fluid at a concentration of 18 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, microfluidic treatment was performed using a domestically produced microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and 2 treatments.

[0045] Example 3: Sodium chloride was added to the virus harvest fluid at a concentration of 30 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, microfluidic treatment was performed using a domestically produced microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and 2 treatments.

[0046] Example 4: Sodium chloride was added to the virus harvest fluid at a concentration of 24 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, microfluidic treatment was performed using a domestically produced microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and 2 treatments.

[0047] Example 5: Sodium chloride was added to the virus harvesting fluid at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, microfluidic treatment was performed using a domestically produced microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and 2 treatments.

[0048] Performance Test 1: Residual DNA testing was performed on Examples 1-5. The methods for detecting residual DNA and DNA removal rate are as follows: The HEK293 DNA quantitative reference standard and DNA diluent from the HEK293 DNA kit were thawed and mixed under ice bath conditions; using the HEK293 DNA quantitative reference standard as the standard, a series of concentration gradient standards of 300 pg / μL, 30 pg / μL, 3 pg / μL, 300 fg / μL, and 30 fg / μL were prepared using DNA diluent to establish a standard curve; after DNA extraction and purification, the adenovirus-containing solution of the test sample was detected using a real-time fluorescence quantitative PCR detection system, and the concentration and removal rate of host residual DNA in the sample were calculated according to the standard curve; subsequent methods for detecting residual DNA and DNA removal rate all refer to this method; as shown in Table 1:

[0049] Table 1: Effects of different sodium chloride concentrations on DNA removal from virus harvest fluid

[0050]

[0051] Result Analysis 1: As shown in Table 1 above, when the concentration of sodium chloride added to the virus harvest fluid is 24~30 g / L, the removal rate of host DNA is excellent; among them, when the concentration of sodium chloride added is 26 g / L, the removal rate of host DNA is the most efficient.

[0052] 3. As shown above, the highest efficiency in removing host DNA is achieved when the sodium chloride concentration in the virus harvesting fluid is 26 g / L. Based on Example 1, sodium chloride was added to each batch of virus harvesting fluid (Example 6) at a concentration of 26 g / L, and treated using an imported microfluidic device. Simultaneously, another portion of virus harvesting fluid was taken from each batch, designated as Comparative Example 1, and sodium chloride was added to it at a concentration of 26 g / L, followed by static ultra-high pressure treatment. This was used to verify the effectiveness of microfluidic treatment and determine the optimal microfluidic device, as detailed below:

[0053] Example 6: Sodium chloride was added to the virus harvesting fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with imported microfluidic equipment. The parameters of the microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and treatment times of 2.

[0054] Comparative Example 1: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours and then subjected to static ultra-high pressure treatment at 4000 bar for 45 minutes.

[0055] Performance Test 2: Residual DNA tests were performed on Example 6 and Comparative Example 1, as detailed in Table 2:

[0056] Table 2: Effects of different equipment / methods on DNA removal from virus harvest fluid

[0057]

[0058] Result Analysis 2: As can be seen from the test results in Table 2 above, microfluidic treatment can achieve efficient removal of host DNA from the virus, especially when using imported microfluidic equipment for microfluidic treatment.

[0059] 4. Based on the test results of Examples 1-6, it can be seen that when the sodium chloride concentration is 26 g / L, microfluidic treatment of the virus harvest fluid using an imported microfluidic device can achieve more efficient removal of host DNA from the virus. Now, based on Example 6, a single sample of virus harvest fluid from each batch was taken and designated as Comparative Example 2. Sodium chloride was added to Comparative Example 2 and to Examples 7-8 of each batch of virus harvest fluid at a concentration of 26 g / L, and then microfluidic treatment was performed using an imported microfluidic device. Specifically, Comparative Example 2 used a Z-shaped channel structure for microfluidic treatment, while Examples 7-8 used 50 μm and 100 μm channel pore sizes, respectively, as detailed below:

[0060] Comparative Example 2: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After complete dissolution, the mixture was shaken for 3 hours and then microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were: Z-shaped channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and 2 treatment cycles.

[0061] Performance Test 3: Residual DNA was tested on Comparative Example 2, as shown in Table 3:

[0062] Table 3: Effect of channel structure type of microfluidic device on DNA removal from virus harvest fluid

[0063]

[0064] Result Analysis 3: As can be seen from the test results in Table 3 above, the microfluidic treatment effect of the Z-type channel structure is lower than that of the Y-type channel structure. The amount of residual DNA after treatment with the Z-type channel structure is more than twice that of the residual DNA after treatment with the Y-type channel structure.

[0065] Example 7: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with imported microfluidic equipment. The parameters of the microfluidic treatment were: Y-type channel structure with a channel diameter of 50 μm; treatment pressure of 3000 bar; and treatment times of 2.

[0066] Example 8: Sodium chloride was added to the virus harvesting fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 100 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0067] Performance Test 4: Residual DNA tests were performed on Examples 7-8, as detailed in Table 4:

[0068] Table 4: Effect of channel aperture size of microfluidic equipment on DNA removal from virus harvest fluid

[0069]

[0070] Result Analysis 4: The test results in Table 4 above show that when the Y-type channel aperture is 25~50μm, microfluidic treatment of the virus harvest fluid exhibits a high host DNA removal effect.

[0071] 5. As shown above, when the Y-channel aperture is 25-50 μm, microfluidic treatment of the virus harvest fluid exhibits a high host DNA removal effect. Based on Example 7, Examples 9-12 employ microfluidic pressures of 2000 bar, 2250 bar, 2500 bar, and 2750 bar respectively to obtain the optimal process parameters for microfluidic treatment, as detailed below:

[0072] Example 9: Sodium chloride was added to the virus harvesting fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2000 bar; and 2 treatments.

[0073] Example 10: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with imported microfluidic equipment. The parameters of the microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2250 bar; and 2 treatments.

[0074] Example 11: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with imported microfluidic equipment. The parameters of the microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2500 bar; and 2 treatments.

[0075] Example 12: Sodium chloride was added to the virus harvest fluid at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with imported microfluidic equipment. The parameters of the microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2750 bar; and 2 treatments.

[0076] Performance Test 5: Residual DNA testing was performed on Examples 9-12. The viral titer was detected as follows: Under ice bath conditions, the adenovirus-containing solution was serially diluted 10-fold to 10^6 using DMEM complete medium as the diluent. -12 The diluted virus solution was added to a 96-well cell culture plate containing 293 cells and cultured at 37°C with 5% CO2 for 10 days. The cytopathic effect was observed daily, and the viral titer of the adenovirus-containing sample was calculated using the half-maximal dose method. Subsequent methods for detecting viral titers followed the same approach. See Table 5 for details.

[0077] Table 5: Effect of microfluidic pressure on DNA removal from virus harvest fluid

[0078]

[0079] Result Analysis 5: The test results in Table 5 above show that when the pressure is ≥2500 bar, the removal effect on host DNA is basically consistent. At 2500~3000 bar, there is no effect on viral titer. (See details...) Figure 2 .

[0080] Performance Test 6: One sample from each batch was randomly selected under a microjets pressure of 2500–3000 bar for further testing. The size of the host DNA fragments in each batch was measured using a DNA fragment analyzer. Subsequent DNA fragment size measurements were performed using the DNA fragment analyzer, as detailed in Table 6.

[0081] Table 6: Size of host DNA fragments in virus harvest fluid samples under microfluidic pressure of 2500–3000 bar

[0082]

[0083] Among them, IG (below the last point from the standard curve): the detected value is 1.00x10 lower than the lowest concentration point of the standard curve. -3 pg / uL.

[0084] Result Analysis 6: The test results in Table 6 above show that when the pressure is ≥2500 bar, the host DNA fragments in the virus harvest fluid are generally less than 200 bp, which meets the relevant regulatory requirements for oncolytic viruses.

[0085] In summary, based on the test results of Examples 1-12 and Comparative Examples 1-2, it can be seen that when the concentration of sodium chloride added to the virus harvesting fluid is 24-30 g / L, the microfluidic treatment adopts a Y-shaped channel structure with a channel pore size of 25-50 μm, and the microfluidic pressure is 2500-3000 bar, the host DNA content can be removed more efficiently.

[0086] II. Treatment of the ultrafiltration concentrate:

[0087] Examples 13-23: A method for preparing low-host DNA in a human adenovirus preparation:

[0088] 1. Obtaining Ultrafiltration Concentrate: The above three batches of virus harvested fluid were subjected to ultrafiltration concentration treatment: First, the virus harvested fluid was filtered using a 0.8 μm filter cartridge, and then filtered again using a 0.65 μm filter cartridge to obtain a clear virus harvested fluid; then, the clear virus harvested fluid was ultrafiltered and concentrated 12 times using a 300 KD low protein adsorption ultrafiltration membrane to obtain a primary concentrate; finally, the primary concentrate was washed with 7 volumes of 0.01 mol / L PBS buffer and 9 volumes of 0.35 mol / L sodium chloride solution to obtain the ultrafiltration concentrate;

[0089] 2. Three batches of ultrafiltration concentrate were obtained according to the above method. Eleven portions were taken from each batch and uniformly numbered as Example 13, Example 14, Example 15...Example 23. Different concentrations of sodium chloride were added to each batch of ultrafiltration concentrate from Examples 13 to 17, and then microfluidic treatment was performed using an imported microfluidic device to verify the role of sodium chloride in the ultrafiltration concentrate and to determine the optimal concentration of sodium chloride to be added. The details are as follows:

[0090] Example 13: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0091] Example 14: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 18 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0092] Example 15: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 30 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0093] Example 16: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 24 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0094] Example 17: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0095] Performance Test 7: Residual DNA tests were performed on Examples 13-17, as detailed in Table 7.

[0096] Table 7: Effects of different sodium chloride concentrations on DNA removal from ultrafiltration concentrate

[0097]

[0098] Results Analysis 7: As shown in Table 7 above, when the concentration of sodium chloride added to the ultrafiltration concentrate is 24~30 g / L, the removal rate of host DNA is excellent; among them, when the concentration of sodium chloride added is 28 g / L, the removal rate of host DNA is the most efficient.

[0099] 3. As shown above, the removal rate of host DNA is most efficient when the sodium chloride concentration in the ultrafiltration concentrate is 28 g / L. Based on Example 13, sodium chloride was added to each batch of ultrafiltration concentrate in Examples 18-23 at a concentration of 28 g / L. Simultaneously, Examples 18-21 were subjected to microjets with pressures of 2000 bar, 2250 bar, 2500 bar, and 2750 bar, respectively, and Examples 22-23 used channel pore sizes of 25 μm and 100 μm, respectively, to obtain the optimal process parameters for microjets, as detailed below:

[0100] Example 18: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 2000 bar; and 2 treatment cycles.

[0101] Example 19: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 2250 bar; and 2 treatment cycles.

[0102] Example 20: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 2500 bar; and 2 treatment cycles.

[0103] Example 21: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 2750 bar; and 2 treatment cycles.

[0104] Performance Test 8: Residual DNA tests were performed on Examples 18-21, as detailed in Table 8:

[0105] Table 8: Effect of microfluidic pressure on DNA removal from ultrafiltration concentrate.

[0106]

[0107] Result Analysis 8: The test results in Table 8 above show that when the pressure is ≥2500 bar, the removal effect of host DNA in the ultrafiltration concentrate is basically consistent. At 2500~3000 bar, there is no effect on the viral titer. (See details in Table 8.) Figure 3 .

[0108] Performance Test 9: One sample from each batch was randomly selected under a microjets pressure of 2500–3000 bar for further testing to determine the size of the host DNA fragment in each batch, as shown in Table 9.

[0109] Table 9: Size of host DNA fragments in ultrafiltration concentrate samples under microfluidic pressure of 2500–3000 bar

[0110]

[0111] Result Analysis 9: The test results in Table 9 above show that when the pressure is ≥2500 bar, the host DNA fragments in the ultrafiltration concentrate are basically all below 200 bp, which meets the relevant regulatory requirements for oncolytic viruses.

[0112] Example 22: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 25 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0113] Example 23: Sodium chloride was added to the ultrafiltration concentrate at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with imported microfluidic equipment. The parameters of the microfluidic treatment were: Y-type channel structure with a channel pore size of 100 μm; treatment pressure of 3000 bar; and treatment times of 2.

[0114] Performance Test 10: Residual DNA tests were performed on Examples 22-23, as detailed in Table 10.

[0115] Table 10: Effect of microfluidic device channel pore size on DNA removal from ultrafiltration concentrate

[0116]

[0117] Results Analysis 10: The test results in Table 10 above show that when the Y-channel pore size is 25~50μm, microfluidic treatment of the ultrafiltration concentrate exhibits a high host DNA removal effect.

[0118] In summary, based on the test results of Examples 13-23, it can be seen that when the concentration of sodium chloride added to the ultrafiltration concentrate is 24-30 g / L, the microfluidic treatment adopts a Y-type channel structure with a channel pore size of 25-50 μm, and the microfluidic pressure is 2500-3000 bar, the host DNA content can be removed more efficiently.

[0119] III. Treatment of the original solution:

[0120] Examples 24-34: A method for preparing low-host DNA in a human adenovirus preparation:

[0121] 1. Obtaining the stock solution: The above three batches of ultrafiltration concentrate were purified by molecular sieve gel chromatography: The ultrafiltration concentrate was separated and purified by molecular sieve gel chromatography at a linear flow rate of 40 cm / h, and eluted with 0.01 mol / L PBS buffer, with the pressure controlled at <0.8 bar during the process. The first peak was collected to obtain the stock solution.

[0122] 2. Three batches of stock solutions were obtained according to the above method. Eleven portions of each batch were taken and uniformly numbered as Example 24, Example 25, Example 26...Example 34. Different concentrations of sodium chloride were added to each batch of stock solutions from Examples 24 to 28. Then, imported microfluidic equipment was used to treat the solutions with microfluidics to verify the role of sodium chloride in the stock solutions and to determine the optimal concentration of sodium chloride to be added. The details are as follows:

[0123] Example 24: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0124] Example 25: Sodium chloride was added to the stock solution at a concentration of 18 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0125] Example 26: Sodium chloride was added to the stock solution at a concentration of 30 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0126] Example 27: Sodium chloride was added to the stock solution at a concentration of 24 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0127] Example 28: Sodium chloride was added to the stock solution at a concentration of 28 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0128] Performance Test 11: Residual DNA tests were performed on Examples 24-28, as detailed in Table 11:

[0129] Table 11: Effects of different concentrations of sodium chloride on DNA removal from the original solution

[0130]

[0131] Results Analysis 11: As shown in Table 11 above, when the concentration of sodium chloride added to the stock solution is 24~30 g / L, the removal rate of host DNA is excellent; among them, when the concentration of sodium chloride added is 26 g / L, the removal rate of host DNA is the most efficient.

[0132] 3. As can be seen from the above, the removal rate of host DNA is most efficient when the concentration of sodium chloride added to the stock solution is 26 g / L. Based on Example 24, sodium chloride was added to each batch of stock solution in Examples 29-32 at a concentration of 26 g / L. Examples 29-32 were subjected to microjets with pressures of 2000 bar, 2250 bar, 2500 bar, and 2750 bar, respectively. Examples 33-34 used channel pore sizes of 25 μm and 100 μm, respectively, to obtain the optimal process parameters for microjets, as detailed below:

[0133] Example 29: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2000 bar; and 2 treatments.

[0134] Example 30: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2250 bar; and 2 treatments.

[0135] Example 31: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 2500 bar; and 2 treatments.

[0136] Example 32: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel diameter of 50 μm; treatment pressure of 2750 bar; and 2 treatments.

[0137] Performance Test 12: Residual DNA tests were performed on Examples 29-32, as detailed in Table 12:

[0138] Table 12: Effect of microfluidic pressure on DNA removal from the original solution using microfluidic equipment

[0139]

[0140] Result Analysis 12: The test results in Table 12 above show that when the pressure is ≥2500 bar, the removal effect of host DNA in the original solution is basically consistent. At 2500~3000 bar, there is no effect on the viral titer. (See details...) Figure 4 .

[0141] Performance Test 13: One sample from each batch was randomly selected under a microjets pressure of 2500–3000 bar for further testing to determine the size of the host DNA fragment in each batch, as shown in Table 13.

[0142] Table 13: Size of host DNA fragments in the original solution samples under microfluidic pressure of 2500~3000 bar

[0143]

[0144] Among them, IG (below the last point from the standard curve): the detected value is 1.00x10 lower than the lowest concentration point of the standard curve. -3 pg / uL.

[0145] Results Analysis 13: The test results in Table 13 above show that when the pressure is ≥2500 bar, the host DNA fragments in the original solution are basically all below 200 bp, which meets the relevant regulations for oncolytic viruses.

[0146] Example 33: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel aperture of 25 μm; treatment pressure of 3000 bar; and treatment times of 2 times.

[0147] Example 34: Sodium chloride was added to the stock solution at a concentration of 26 g / L. After it was fully dissolved, it was shaken for 3 hours. Then, it was treated with microfluidic equipment using imported microfluidic equipment. The parameters for microfluidic treatment were: Y-type channel structure with a channel diameter of 100 μm; treatment pressure of 3000 bar; and 2 treatments.

[0148] Performance Test 14: Residual DNA tests were performed on Examples 33-34, as detailed in Table 14.

[0149] Table 14: Effect of microfluidic device channel aperture size on DNA removal from the original solution

[0150]

[0151] Results Analysis 14: The test results in Table 14 above show that when the Y-channel aperture is 25~50μm, microfluidic treatment of the original solution exhibits a high host DNA removal effect.

[0152] In summary, based on the test results of Examples 24-34, it can be seen that when the concentration of sodium chloride added in the stock solution is 24-30 g / L, the microfluidic treatment adopts a Y-shaped channel structure with a channel pore size of 25-50 μm, and the microfluidic pressure is 2500-3000 bar, the host DNA content can be removed more efficiently.

[0153] IV. Examples 35-46:

[0154] Based on the foregoing, it is known that when the concentration of sodium chloride added to the virus harvest fluid, ultrafiltration concentrate, or stock solution is 24-30 g / L, and the microfluidic treatment employs a Y-shaped channel structure with a channel pore size of 25-50 μm and a microfluidic pressure of 2500-3000 bar, the removal of host DNA can be more efficient. In this invention, potassium chloride, arginine hydrochloride, a nonionic surfactant, and PBS buffer are further added to the pretreatment agent to improve the removal efficiency of host DNA. Specifically:

[0155] 1. Take four portions from each of the above three batches of virus harvest fluid, ultrafiltration concentrate, and stock solution, and uniformly number them as Example 35, Example 36, Example 37...Example 46; wherein, Examples 35-38 are virus harvest fluid, Examples 39-42 are ultrafiltration concentrate, and Examples 43-46 are stock solutions; add a pretreatment agent to them and perform microfluidic treatment, as follows:

[0156] Example 35: Sodium chloride (26 g / L), arginine hydrochloride (2 g / L), Tween 20 (0.1 mL / L), and potassium chloride (5 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0157] Example 36: Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0158] Example 37: Sodium chloride (26 g / L), arginine hydrochloride (4 g / L), Tween 20 (0.6 mL / L), and potassium chloride (8 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0159] Example 38: Sodium chloride (26 g / L), arginine hydrochloride (5 g / L), Tween 20 (1 mL / L), and potassium chloride (10 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0160] Example 39: Sodium chloride (28 g / L), arginine hydrochloride (2 g / L), Tween 20 (0.1 mL / L), and potassium chloride (5 g / L) were added to the ultrafiltration concentrate. PBS buffer was added simultaneously to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0161] Example 40: Sodium chloride (28 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the ultrafiltration concentrate. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0162] Example 41: Sodium chloride (28 g / L), arginine hydrochloride (4 g / L), Tween 20 (0.6 mL / L), and potassium chloride (8 g / L) were added to the ultrafiltration concentrate. PBS buffer was added simultaneously to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0163] Example 42: Sodium chloride (28 g / L), arginine hydrochloride (5 g / L), Tween 20 (1 mL / L), and potassium chloride (10 g / L) were added to the ultrafiltration concentrate. PBS buffer was added simultaneously to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were: Y-type channel structure with a pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0164] Example 43: Sodium chloride (26 g / L), arginine hydrochloride (2 g / L), Tween 20 (0.1 mL / L), and potassium chloride (5 g / L) were added to the stock solution. PBS buffer was added to stabilize the pH, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The microfluidic treatment parameters were: Y-type channel structure with a pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0165] Example 44: Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the stock solution. PBS buffer was added to stabilize the pH, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0166] Example 45: Sodium chloride (26 g / L), arginine hydrochloride (4 g / L), Tween 20 (0.6 mL / L), and potassium chloride (8 g / L) were added to the stock solution. PBS buffer was added to stabilize the pH, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The microfluidic treatment parameters were: Y-channel structure with a pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0167] Example 46: Sodium chloride (26 g / L), arginine hydrochloride (5 g / L), Tween 20 (1 mL / L), and potassium chloride (10 g / L) were added to the stock solution. PBS buffer was added to stabilize the pH, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0168] Performance Test 15: Residual DNA tests were performed on Examples 35-46, as detailed in Tables 15-17:

[0169] Table 15: Effects of different concentrations of pretreatment agents on DNA removal from virus harvest fluid

[0170]

[0171] Table 16: Effects of different concentrations of pretreatment agents on DNA removal from ultrafiltration concentrate

[0172]

[0173] Table 17: Effects of different concentrations of pretreatment agents on DNA removal from the stock solution

[0174]

[0175] Results Analysis 15: The test results in Tables 15-17 above show that potassium chloride, arginine hydrochloride, and nonionic surfactant (Tween 20) have a significant synergistic promoting effect on sodium chloride. After the addition of these substances, the removal effect of host DNA in virus harvest fluid, ultrafiltration concentrate, and stock solution is significantly enhanced.

[0176] 2. To verify the structure-activity relationship of potassium chloride, arginine hydrochloride, and nonionic surfactant (Tween 20) on sodium chloride, comparative examples 3-13 were further set up based on Example 36, as follows:

[0177] Comparative Example 3: Comparative Example 3 is based on Example 36, with the following adjustments: arginine hydrochloride and PBS buffer are not added, while other processes remain unchanged. Specifically:

[0178] Sodium chloride (26 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. After thorough dissolution, the mixture was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were as follows: Y-type channel structure with a channel aperture of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0179] Comparative Example 4: Comparative Example 4 is based on Example 36, with the following adjustment: PBS buffer is not added, while other processes remain unchanged. Specifically:

[0180] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. After thorough dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The parameters for microfluidic treatment were as follows: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0181] Comparative Example 5: Comparative Example 5 is based on Example 36, with the following adjustment: no nonionic surfactant is added, while other processes remain unchanged. Specifically:

[0182] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0183] Comparative Example 6: Comparative Example 6 is based on Example 36, with the following adjustment: potassium chloride is not added, while other processes remain unchanged. Specifically:

[0184] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), and Tween 20 (0.4 mL / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0185] Comparative Example 7: Comparative Example 7 is based on Example 36, with the following adjustment: no pretreatment agent is added, while other processes remain unchanged. Specifically:

[0186] Imported microfluidic equipment was used to treat the virus harvest fluid. The parameters for microfluidic treatment were as follows: Y-shaped channel structure with a channel diameter of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0187] Comparative Example 8: Comparative Example 8 is based on Example 36, with the following adjustment: the concentration of arginine hydrochloride added is 1 g / L, while other processes remain unchanged. Specifically:

[0188] Sodium chloride (26 g / L), arginine hydrochloride (1 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0189] Comparative Example 9: Comparative Example 9 is based on Example 36, with the following adjustment: the concentration of arginine hydrochloride added is 10 g / L, while other processes remain unchanged. Specifically:

[0190] Sodium chloride (26 g / L), arginine hydrochloride (10 g / L), Tween 20 (0.4 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The microfluidic treatment parameters were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0191] Comparative Example 10: Comparative Example 10 is based on Example 36, with the following adjustment: the concentration of Tween 20 added is 0.01 mL / L, while other processes remain unchanged. Specifically:

[0192] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.01 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0193] Comparative Example 11: Comparative Example 11 is based on Example 36, except that the concentration of Tween 20 was adjusted to 1.5 mL / L, while other processes remained unchanged. Specifically:

[0194] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (1.5 mL / L), and potassium chloride (7 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0195] Comparative Example 12: Comparative Example 12 is based on Example 36, with the following adjustment: the concentration of potassium chloride added is 1 g / L, while other processes remain unchanged. Specifically:

[0196] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.4 mL / L), and potassium chloride (1 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, microfluidic treatment was performed using an imported microfluidic device. The microfluidic treatment parameters were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0197] Comparative Example 13: Comparative Example 13 is based on Example 36, with the following adjustment: the concentration of potassium chloride added is 15 g / L, while other processes remain unchanged. Specifically:

[0198] Sodium chloride (26 g / L), arginine hydrochloride (3 g / L), Tween 20 (0.4 mL / L), and potassium chloride (15 g / L) were added to the virus harvest fluid. PBS buffer was added to stabilize the pH of the solution, ensuring the pH variation did not exceed ±0.2. After complete dissolution, the solution was shaken for 3 hours. Then, an imported microfluidic device was used for microfluidic treatment. The parameters for microfluidic treatment were: Y-type channel structure with a channel pore size of 50 μm; treatment pressure of 3000 bar; and two treatment cycles.

[0199] Performance Test 16: Residual DNA was tested on comparative examples 3-13, as shown in Table 18.

[0200] Table 18: Effects of relevant pretreatment agent components on DNA removal from virus harvest fluid

[0201]

[0202] Result Analysis 16: The components in the pretreatment agent of this invention work closely together to enhance the efficiency of host DNA removal.

[0203] In summary, this invention achieves highly efficient removal of host DNA and efficient virus recovery through the synergistic effect of the components in the pretreatment agent and the control of process parameters in microfluidic treatment. Studies have found that microfluidic treatment at 2500-3000 bar achieves a host DNA removal rate of 90-99%, with residual host DNA levels ≤1 ng / dose, far below the standards stipulated in the National Pharmacopoeia, and residual fragments ≤200 bp. The product quality exhibits high stability and high controllability.

[0204] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for preparing a human adenovirus preparation with low host DNA, characterized in that: This is achieved by adding a pretreatment agent to the adenovirus-containing solution, pretreating it, and then subjecting it to microfluidic treatment. The pretreatment agents include sodium chloride, arginine hydrochloride, PBS buffer, Tween 20, and potassium chloride; per liter of adenovirus-containing solution, the concentration of sodium chloride is 24-30 g / L, the concentration of arginine hydrochloride is 3-4 g / L, the concentration of Tween 20 is 0.4-0.6 mL / L, the concentration of potassium chloride is 7-8 g / L, and the PBS buffer is used to stabilize the pH. The pressure of the microjet treatment is 2500~3000 bar; The microjet processing adopts a Y-shaped channel structure with a channel aperture of 25~50μm.

2. The method for preparing a human adenovirus preparation with low host DNA according to claim 1, characterized in that: The pretreatment is an oscillation process, and the processing time is 2-4 hours.

3. The method for preparing a human adenovirus preparation with low host DNA according to claim 1, characterized in that: The adenovirus-containing solution is a virus harvest solution, an ultrafiltration concentrate, or a stock solution.

4. The low-host DNA adenovirus preparation obtained by the preparation method of the low-host DNA human adenovirus preparation according to any one of claims 1 to 3.

Images