Polydeoxyribonucleotide inactivation process and application

Abstract

The invention belongs to the technical field of inactivation processes, and particularly relates to a polydeoxyribonucleotide inactivation process and application. According to the inactivation process, the PDRN inactivation liquid is obtained through deep filtration pretreatment, dispersion of acid pickling diatomite and a nano cellulose filter aid in a specific ratio under a specific dissolved oxygen condition, gradient pressure plate-and-frame pressure filtration, ceramic membrane filtration, ultrafiltration and constant-volume percolation, and UV inactivation according to a specific wavelength ratio and dosage. According to the PDRN inactivation liquid prepared through the inactivation technology, the DNA content reaches up to 99% or above, the protein content is lower than 0.2%, the endotoxin content is smaller than 0.005 EU / mL, efficient microorganism inactivation is achieved, meanwhile, PDRN active ingredients are reserved to the maximum extent, the product purity and safety are remarkably improved, and the PDRN inactivation liquid has wide application prospects in the fields of medicine, medical beauty and biomedicine.

Description

Technical Field

[0001] The invention belongs to the technical field of inactivation processes, and in particular relates to a polydeoxyribonucleotide inactivation process and application. Background Art

[0002] Polydeoxyribonucleotide (PDRN), as a functional DNA fragment, has become the core raw material for tissue regeneration therapy in the medical field, skin repair products in the medical beauty industry, and biomedical material development due to its significant biological properties such as promoting cell repair, anti-inflammation, and improving tissue microenvironment.

[0003] However, its industrial preparation process still faces multiple technical bottlenecks: on the one hand, the extraction of PDRN raw materials and subsequent processing links are very likely to introduce contamination from microorganisms such as bacteria and viruses. If there are defects in the inactivation process, the residual endotoxins will trigger the human immune response, seriously threatening product safety; on the other hand, the biological activity of PDRN is highly dependent on its complete nucleotide sequence and spatial conformation. Traditional inactivation methods such as high-temperature sterilization and treatment with strong chemical reagents can easily cause DNA chain breakage, base oxidation, or form irreversible bonds with impurities, leading to the loss of active ingredients and significantly reducing product efficacy.

[0004] In existing technologies, a single inactivation strategy struggles to overcome the dilemma of balancing safety and activity. For example, while high-pressure steam sterilization can effectively kill microorganisms, the high temperature denatures the molecular structure of PDRN. While low-intensity ultraviolet (UV) irradiation can reduce damage to active ingredients, it struggles to completely inactivate photostable microorganisms. Traditional filtration processes often result in nonspecific adsorption of PDRN on the filter surface due to improper filter material selection or extensive parameter control, or lead to residual impurities such as proteins and endotoxins, which in turn lead to problems such as poor process stability and significant batch-to-batch quality fluctuations. Furthermore, the lack of systematic collaborative optimization between the various process links makes it impossible to form an integrated solution that combines efficient sterilization, activity protection, and precise impurity removal.

[0005] Therefore, how to develop a synergistic process system that can precisely control the inactivation intensity, maximize the retention of PDRN biological activity, and achieve deep removal of impurities has become a key technical problem restricting the industrial application of PDRN, and is also an important research direction in the current biomedicine field and inactivation process technology field. Summary of the Invention

[0006] In view of the above-mentioned shortcomings, the present invention provides a polydeoxyribonucleotide inactivation process and application.

[0007] The technical solution of the present invention is: In one aspect, the present invention provides a process for inactivating polydeoxyribonucleotides, comprising the following steps: S1. Pretreatment: Filter the polydeoxyribonucleotide solution through a deep filter and collect the filtered liquid as the coarse filtrate; S2. Filter aid dispersion: Add 0.50-0.55% w / v acid-washed diatomaceous earth and 0.40-0.45% w / v nanocellulose to the coarse filtrate, stir and disperse them evenly under the condition of dissolved oxygen of 0.3-0.5 ppm; S3. Filter pressing: Use a plate and frame filter press to filter, pre-coat the filter cloth with acid-washed diatomaceous earth suspension, and filter at 0.05-0.08Mpa until the flow rate stabilizes to 45-50L / (m 2 h); adjust the pressure to 0.10-0.15 MPa, and linearly increase the pressure to 0.30-0.35 MPa at a rate of 0.05 MPa / min; maintain the pressure at 0.30-0.35 MPa for 20-30 min, and then reduce the pressure to 0.05-0.20 MPa until no droplets are left; and obtain the filtrate; S4, ceramic membrane filtration: using a ceramic membrane to filter the filtrate to obtain a primary filtrate; S5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 30-50 kDa ultrafiltration membrane. When the volume of the retentate is concentrated to 1 / 2 of the original volume of the primary filtrate, the filtration mode is switched to constant volume diafiltration: physiological saline is added at a rate of 2.5-3.0 L / min, and the permeate is discharged at the same rate, displacing a cumulative volume of 3-5 times the original volume of the primary filtrate. When the conductivity is ≤1.5 mS / cm and UV280 is ≤0.02, the diafiltration is stopped and the retentate is further concentrated to 1 / 4-1 / 5 of the original volume of the primary filtrate. The retentate is finally pre-filtered through the membrane to obtain the ultrafiltrate. S6, UV inactivation: A medium-pressure mercury lamp array is installed in the cavity to pass the ultrafiltrate through the cavity, with a cumulative irradiation dose of 20-30 kJ / m 2 ; In the medium pressure mercury lamp array, 185nm accounts for 10-15% and 254nm accounts for 85-90%; S7. Subpackaging: The inactivated solution is subpacked into sterile containers through a sterile filter to obtain a polydeoxyribonucleotide inactivated solution.

[0008] Specifically, the depth filter in step S1 is a 5-20 μm depth filter.

[0009] Preferably, the depth filter in step S1 is a 10 μm depth filter.

[0010] Specifically, the filtration flow rate in step S1 is 50-100 L / h.

[0011] Preferably, the flow rate of the filtration in step S1 is 70-75 L / h.

[0012] Specifically, the amount of the acid-washed diatomaceous earth added in step S2 is 0.50-0.51% w / v, 0.51-0.52% w / v, 0.52-0.53% w / v, 0.53-0.54% w / v or 0.54-0.55% w / v.

[0013] Preferably, the amount of the acid-washed diatomaceous earth added in step S2 is 0.50% w / v or 0.55% w / v.

[0014] Specifically, the amount of nanocellulose added in step S2 is 0.40-0.41% w / v, 0.41-0.42% w / v, 0.42-0.43% w / v, 0.43-0.44% w / v or 0.44-0.45% w / v.

[0015] Preferably, the amount of the acid-washed diatomaceous earth added in step S2 is 0.40% w / v or 0.50% w / v.

[0016] Specifically, the material of the filter cloth in step S3 includes: any one or more of polypropylene, polyester fiber, polyamide, polyvinyl formal fiber, and polytetrafluoroethylene.

[0017] Preferably, the filter cloth in step S3 is made of polypropylene.

[0018] Specifically, the pore size of the filter cloth in step S3 is 5-10 μm.

[0019] Preferably, the pore size of the filter cloth in step S3 is 5 μm.

[0020] Specifically, the pore size of the ceramic membrane in step S4 is 100-200 nm.

[0021] Preferably, the pore size of the ceramic membrane in step S4 is 100 nm or 200 nm.

[0022] Specifically, the membrane flux of the ceramic membrane filtration in step S4 is 100-120LMH.

[0023] Preferably, the membrane flux of the ceramic membrane filtration in step S4 is 100-110LMH or 110-120LMH.

[0024] Further preferably, the membrane flux of the ceramic membrane filtration in step S4 is 100LMH or 120LMH.

[0025] Specifically, the inlet pressure of the ceramic membrane filtration in step S4 is 0.16-0.20 MPa; the outlet pressure is 0.08-0.10 MPa.

[0026] Preferably, the inlet pressure of the ceramic membrane filtration in step S4 is 0.16-0.17 MPa, 0.17-0.18 MPa, 0.18-0.19 MPa or 0.19-0.20 MPa.

[0027] Preferably, the outlet pressure of the ceramic membrane filtration in step S4 is 0.08-0.09 MPa or 0.09-0.10 MPa.

[0028] Specifically, the inlet pressure of the ultrafiltration in step S5 is 0.1-0.2 MPa, and the outlet pressure is 0.05-0.1 MPa.

[0029] Preferably, the inlet pressure of the ultrafiltration in step S5 is 0.2 MPa, and the outlet pressure is 0.1 MPa.

[0030] Specifically, the filter membrane in step S5 is a 0.45-1.0 μm filter membrane.

[0031] Preferably, the filter membrane in step S5 is a 0.45 μm filter membrane.

[0032] Specifically, the sterile filter in step S7 is a 0.1-0.22 μm sterile filter.

[0033] Preferably, the sterile filter in step S7 is a 0.22 μm sterile filter.

[0034] In another aspect, the present invention provides use of any of the above-mentioned inactivation processes in inactivating polydeoxyribonucleotides.

[0035] The beneficial effects of the present invention are: The PDRN inactivation liquid prepared by the inactivation process of the present invention has a DNA content of over 99%, a protein content of less than 0.2%, and an endotoxin content of less than 0.005EU / mL. While achieving efficient microbial inactivation, it retains the PDRN active ingredients to the maximum extent, significantly improving the product purity and safety, and has broad application prospects in the fields of medicine, medical aesthetics, and biomedicine. DETAILED DESCRIPTION

[0036] The present invention will be further clarified and fully illustrated below through examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely illustrative. The experimental methods used in the following examples are routine unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. The polydeoxyribonucleotides used in the present invention were produced by Hengyu Biopharmaceuticals (Shandong) Co., Ltd.

[0037] Example 1 Accurately weigh PDRN and place it in a beaker. Add physiological saline to it, heat in a 40°C water bath and stir until the PDRN is dissolved to obtain a 10 mg / mL polydeoxyribonucleotide solution.

[0038] 1. Pretreatment: Pass the polydeoxyribonucleotide solution through a 10 μm depth filter, control the pH to 7.0-7.5, the temperature to 40°C, and the flow rate to 75 L / h, and collect the filtered liquid as the crude filtrate.

[0039] 2. Dispersion of filter aids: Add 0.55% w / v acid-washed diatomaceous earth and 0.45% w / v nanocellulose to the coarse filtrate, introduce CO2 to make the dissolved oxygen content 0.3 ppm, and stir at 150 rpm for 20 minutes to make it evenly dispersed.

[0040] 3. Filter pressing: Use a plate and frame filter press (filter cloth material: polypropylene, pore size 5μm) to filter, pre-coat the filter cloth with 0.2% w / v acid-washed diatomaceous earth suspension to form a 1-2mm thin layer, and filter at 0.08Mpa until the flow rate stabilizes to 50L / (m 2 ·h); adjust the pressure to 0.15 MPa, and increase it linearly to 0.35 MPa at a rate of 0.05 MPa / min; maintain it at 0.35 MPa for 20 min, then reduce the pressure to 0.10 MPa until there is no droplet, and obtain the filtrate.

[0041] 4. Ceramic membrane filtration: A ceramic membrane with a pore size of 200 nm is used, the inlet pressure is controlled at 0.16 MPa, the outlet pressure is controlled at 0.08 MPa, the membrane flux is 100 LMH, and the filtrate is filtered to obtain a primary filtrate.

[0042] 5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 50kDa ultrafiltration membrane, with the inlet pressure controlled at 0.2MPa and the outlet pressure at 0.1MPa. The circulation flow rate is adjusted to 5m / s and the membrane flux is 75LMH. When the retentate volume is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to constant volume diafiltration mode: add 37°C normal saline at a rate of 2.5L / min while discharging the permeate at the same rate, displacing a cumulative volume of three times the original volume of the primary filtrate. Sample the permeate every 30 minutes for testing. When the conductivity is ≤1.5mS / cm and the UV280 is ≤0.02, stop diafiltration and continue concentrating the retentate to 1 / 4 of the original volume of the primary filtrate. The retentate is finally pre-filtered through a 0.45μm filter membrane to obtain the ultrafiltrate.

[0043] 6. UV inactivation: A medium-pressure mercury lamp array (15% at 185nm and 85% at 254nm) is installed in the cavity to allow the ultrafiltrate to pass through the cavity, with a cumulative irradiation dose of 30kJ / m 2 .

[0044] 7. Packaging: After inactivation, the solution is dispensed into sterile containers through a sterile filter (0.22 μm) to obtain the PDRN inactivated solution.

[0045] Example 2 Accurately weigh PDRN and place it in a beaker. Add physiological saline to it, heat in a 40°C water bath and stir until the PDRN is dissolved to obtain a 10 mg / mL polydeoxyribonucleotide solution.

[0046] 1. Pretreatment: Pass the polydeoxyribonucleotide solution through a 10 μm depth filter, control the pH to 7.0-7.5, the temperature to 40°C, and the flow rate to 70 L / h, and collect the filtered liquid as the crude filtrate.

[0047] 2. Dispersion of filter aids: Add 0.50% w / v acid-washed diatomaceous earth and 0.40% w / v nanocellulose to the coarse filtrate, introduce CO2 to make the dissolved oxygen content 0.5 ppm, and stir at 150 rpm for 20 minutes to make it evenly dispersed.

[0048] 3. Filter pressing: Use a plate and frame filter press (filter cloth material: polypropylene, pore size 5μm) to filter, pre-coat the filter cloth with 0.4% w / v acid-washed diatomaceous earth suspension to form a 1-2mm thin layer, and filter at 0.08Mpa until the flow rate stabilizes to 45L / (m 2 ·h); adjust the pressure to 0.10 MPa, and increase it linearly to 0.30 MPa at a rate of 0.05 MPa / min; maintain it at 0.30 MPa for 30 min, then reduce the pressure to 0.10 MPa until there is no droplet, and obtain the filtrate.

[0049] 4. Ceramic membrane filtration: A ceramic membrane with a pore size of 100 nm is used, the inlet pressure is controlled at 0.2 MPa, the outlet pressure is controlled at 0.1 MPa, the membrane flux is 120 LMH, and the filtrate is filtered to obtain a primary filtrate.

[0050] 5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 30kDa ultrafiltration membrane, with the inlet pressure controlled at 0.2MPa and the outlet pressure at 0.1MPa. The circulation flow rate is adjusted to 5m / s and the membrane flux is 50LMH. When the retentate volume is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to constant volume diafiltration mode: add 37°C normal saline at a rate of 3.0L / min while discharging the permeate at the same rate, displacing a cumulative volume of 5 times the original volume of the primary filtrate. Sample the permeate every 30 minutes for testing. When the conductivity is ≤1.5mS / cm and the UV280 is ≤0.02, stop diafiltration and continue concentrating the retentate to 1 / 5 of the original volume of the primary filtrate. The retentate is finally pre-filtered through a 0.45μm filter membrane to obtain the ultrafiltrate.

[0051] 6. UV inactivation: A medium-pressure mercury lamp array (10% at 185nm and 90% at 254nm) is installed in the cavity to allow the ultrafiltrate to pass through the cavity, with a cumulative irradiation dose of 20kJ / m 2 .

[0052] 7. Packaging: After inactivation, the solution is dispensed into sterile containers through a sterile filter (0.22 μm) to obtain the PDRN inactivated solution.

[0053] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is only that "2. Dispersion of filter aid" is different. The specific steps are: 2. Dispersion of filter aids: Add 0.45% w / v acid-washed diatomaceous earth and 0.55% w / v nanocellulose to the coarse filtrate, introduce CO2 to make the dissolved oxygen content 0.7 ppm, and stir at 150 rpm for 20 minutes to make it evenly dispersed.

[0054] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is only that "2. Dispersion of filter aid" is different. The specific steps are: 2. Dispersion of filter aids: Add 0.60% w / v acid-washed diatomaceous earth and 0.40% w / v nanocellulose to the coarse filtrate, introduce CO2 to make the dissolved oxygen content 0.2 ppm, and stir at 150 rpm for 20 minutes to make it evenly dispersed.

[0055] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is only that: "3. Filter Press" is different, and the specific steps are: 3. Filter pressing: Use a plate and frame filter press (filter cloth material: polypropylene, pore size 5μm) for filter pressing, pre-coat the filter cloth with 0.2% w / v acid-washed diatomaceous earth suspension to form a 1-2mm thin layer, and filter at 0.05Mpa until the flow rate stabilizes to 50L / (m 2 ·h); adjust the pressure to 0.10 MPa, and linearly increase the pressure to 0.50 MPa at a rate of 0.05 MPa / min; maintain 0.50 MPa for 20 min, and then reduce the pressure to 0.10 MPa until no droplets are left to obtain the filtrate.

[0056] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is only that: "3. Filter pressing" is different, and the specific steps are: 3. Filter pressing: Use a plate and frame filter press (filter cloth material: polypropylene, pore size 5μm) for filter pressing, pre-coat the filter cloth with 0.2% w / v acid-washed diatomaceous earth suspension to form a 1-2mm thin layer, and filter at 0.08Mpa until the flow rate stabilizes to 40L / (m 2·h); adjust the pressure to 0.20 MPa, and increase it linearly to 0.35 MPa at a rate of 0.05 MPa / min; maintain it at 0.35 MPa for 40 min, then reduce the pressure to 0.10 MPa until there is no droplet, and obtain the filtrate.

[0057] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is only that: "5. Ultrafiltration" is different, and the specific steps are: 5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 50kDa ultrafiltration membrane, with the inlet pressure controlled at 0.2MPa and the outlet pressure at 0.1MPa. The circulation flow rate is adjusted to 5m / s and the membrane flux is 75LMH. When the retentate volume is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to constant volume diafiltration mode: add 37°C normal saline at a rate of 2.0L / min while discharging the permeate at the same rate, displacing a cumulative volume of twice the original volume of the primary filtrate. Sample the permeate every 30 minutes for testing. When the conductivity is ≤1.5mS / cm and the UV280 is ≤0.02, stop diafiltration and continue concentrating the retentate to 1 / 3 of the original volume of the primary filtrate. The retentate is finally pre-filtered through a 0.45μm filter membrane to obtain the ultrafiltrate.

[0058] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is only that: "5. Ultrafiltration" is different, and the specific steps are: 5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 50kDa ultrafiltration membrane, with the inlet pressure controlled at 0.2MPa and the outlet pressure at 0.1MPa. The circulation flow rate is adjusted to 5m / s and the membrane flux is 75LMH. When the retentate volume is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to constant volume diafiltration mode: add 37°C normal saline at a rate of 4.0L / min while discharging the permeate at the same rate, displacing a cumulative 8 times the original volume of the primary filtrate. Sample the permeate every 30 minutes for testing. When the conductivity is ≤1.5mS / cm and the UV280 is ≤0.02, stop diafiltration and continue concentrating the retentate to 1 / 4 of the original volume of the primary filtrate. The retentate is finally pre-filtered through a 0.45μm filter membrane to obtain the ultrafiltrate.

[0059] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is only that: "2. Dispersion of filter aid", "3. Press filtration" and "5. Ultrafiltration" are different. The specific steps are: 2. Dispersion of filter aids: Add 0.45% w / v acid-washed diatomaceous earth and 0.55% w / v nanocellulose to the coarse filtrate, introduce CO2 to make the dissolved oxygen content 0.7 ppm, and stir at 150 rpm for 20 minutes to make it evenly dispersed.

[0060] 3. Filter pressing: Use a plate and frame filter press (filter cloth material: polypropylene, pore size 5μm) to filter, pre-coat the filter cloth with 0.2% w / v acid-washed diatomaceous earth suspension to form a 1-2mm thin layer, and filter at 0.08Mpa until the flow rate stabilizes to 50L / (m 2 ·h); adjust the pressure to 0.20 MPa, and increase it linearly to 0.50 MPa at a rate of 0.05 MPa / min; maintain it at 0.50 MPa for 20 min, then reduce the pressure to 0.10 MPa until there is no droplet, and obtain the filtrate.

[0061] 5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 50kDa ultrafiltration membrane, with the inlet pressure controlled at 0.2MPa and the outlet pressure at 0.1MPa. The circulation flow rate is adjusted to 5m / s and the membrane flux is 75LMH. When the retentate volume is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to constant volume diafiltration mode: add 37°C normal saline at a rate of 2.0L / min while discharging the permeate at the same rate, displacing a cumulative volume of twice the original volume of the primary filtrate. Sample the permeate every 30 minutes for testing. When the conductivity is ≤1.5mS / cm and the UV280 is ≤0.02, stop diafiltration and continue concentrating the retentate to 1 / 3 of the original volume of the primary filtrate. The retentate is finally pre-filtered through a 0.45μm filter membrane to obtain the ultrafiltrate.

[0062] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is only that: "6. UV inactivation" is different, and the specific steps are: 6. UV inactivation: A medium-pressure mercury lamp array (185nm accounts for 20%, 254nm accounts for 80%) is installed in the cavity to allow the ultrafiltrate to pass through the cavity, with a cumulative irradiation dose of 50kJ / m 2 .

[0063] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is only that: "2. Dispersion of filter aid" and "6. UV inactivation" are different. The specific steps are: 2. Dispersion of filter aids: Add 0.45% w / v acid-washed diatomaceous earth and 0.55% w / v nanocellulose to the coarse filtrate, introduce CO2 to make the dissolved oxygen content 0.7 ppm, and stir at 150 rpm for 20 minutes to make it evenly dispersed.

[0064] 6. UV inactivation: A medium-pressure mercury lamp array (185nm accounts for 20%, 254nm accounts for 80%) is installed in the cavity to allow the ultrafiltrate to pass through the cavity, with a cumulative irradiation dose of 50kJ / m 2 .

[0065] Experimental Example 1 Determination of DNA content in PDRN inactivation solution Take the PDRN inactivation solution of Example 1-Example 2, Comparative Example 1-Comparative Example 9, and appropriately dilute it with normal saline so that its absorbance is within the linear range of the standard curve. Using normal saline as a blank, the absorbance of the PDRN inactivation solution is measured at 260nm. DNA concentration in the PDRN inactivation solution is calculated according to absorbance. The measurement results are shown in Table 1: Table 1 DNA content determination

[0066] The above results show that the DNA content in the PDRN inactivation solution prepared by the inactivation process of Example 1-Example 2 is as high as more than 99%. Comparative Example 1-Comparative Example 9 all result in a decrease in DNA content compared to the single or composite process adjustment of Example 1. The above results show that under the specific acid-washed diatomaceous earth and nanocellulose dosage, specific dissolved oxygen content, specific filter press and ultrafiltration conditions, and specific UV inactivation conditions of the present invention, it is possible to ensure that the PDRN active ingredient is retained to the greatest extent under the premise of microbial inactivation.

[0067] Comparative Example 9 differs from Example 1 solely in the filter aid dispersion and UV inactivation procedures. Comparative Example 1 differs from Example 1 solely in the filter aid dispersion procedure. Comparative Example 8 differs from Example 1 solely in the UV inactivation procedure. Using Comparative Example 9 as a comparison, the synergistic effect of the filter aid dispersion and UV inactivation procedures in the inactivation process of the present invention was evaluated.

[0068] Compared with Comparative Example 9, the DNA content in the PDRN inactivation solution of Comparative Example 1 increased by 3.78%, the DNA content in the PDRN inactivation solution of Comparative Example 2 increased by 0.53%, and the DNA content in Example 1 increased by 4.93%. It can be seen that the inactivation process of the present invention combines the "filter aid dispersion" process and the "UV inactivation" process to achieve a synergistic effect of 1+1>2 (4.93%>3.78%+0.53%).

[0069] Experimental Example 2 Determination of protein content in PDRN inactivated solution The PDRN inactivation solutions of Examples 1-2 and Comparative Examples 1-9 were appropriately diluted with physiological saline so that their absorbance was within the linear range of the standard curve. The protein content in the PDRN inactivation solution was determined using a protein concentration assay kit (Sangon Biotech, C504031-1000). The results are shown in Table 2: Table 2 Protein content determination

[0070] The above results show that the protein content in the PDRN inactivated solution prepared by the inactivation process of Example 1-Example 2 is below 0.20%. It shows that precise control of the inactivation process steps of the present invention is the key to reducing the protein content of the PDRN inactivated solution and improving product purity.

[0071] Experimental Example 3 PDRN inactivation solution activity determination 1. Adjust the HUVEC cell density to 1.25×10 5 1.25×10 cells / ml; add 200 μL of 1.25×10 5 Cells / ml were placed in a 37°C incubator and incubated overnight; 2. Discard all cell supernatant, add 200 μL of EBM2 medium containing 0.5% FBS, and incubate in a 37°C incubator for 24 hours; 3. Discard 100 μL of cell supernatant, create wounds on HUVEC cells using a scratcher, and wash twice with serum-free EBM2 medium; 4. Place the cells in a living cell chamber and add 100 µg / mL of the PDRN inactivation solution from Examples 1-2 and Comparative Examples 1-9 diluted in complete culture medium (EBM2 cell culture medium containing 0.5% FBS and 1% penicillin / streptomycin). Complete culture medium served as a control group. After 24 hours, photographs were taken and the cell migration area was analyzed using ImageJ. The cell migration rate was calculated. The results are shown in Table 3.

[0072] Table 3 Cell migration rate determination

[0073] The PDRN inactivated solution prepared by the inactivation process of Examples 1 and 2 has biological activity that promotes cell migration. This further demonstrates that the inactivation process of the present invention, through multi-step collaborative optimization, maximizes the biological activity of PDRN while ensuring high purity (DNA>99%, protein<0.2%).

[0074] Experimental Example 4 Determination of endotoxin content in PDRN inactivated solution Endotoxin detection was performed according to the photometric method in General Chapter 1143 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The results are shown in Table 4: Table 4 Endotoxin content determination

[0075] The above results show that the endotoxin content of the PDRN inactivation solution of Example 1-Example 2 is less than 0.005EU / mL, which meets the requirements.

[0076] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A process for inactivating polydeoxyribonucleotides, characterized in that: Described inactivation process comprises the following steps: S1. Pretreatment: Filter the polydeoxyribonucleotide solution through a deep filter and collect the filtered liquid as the coarse filtrate; S2. Filter aid dispersion: Add 0.50-0.55% w / v acid-washed diatomaceous earth and 0.40-0.45% w / v nanocellulose to the coarse filtrate, stir and disperse them evenly under the condition of dissolved oxygen of 0.3-0.5 ppm; S3. Filter pressing: Use a plate and frame filter press to filter, pre-coat the filter cloth with acid-washed diatomaceous earth suspension, and filter at 0.05-0.08Mpa until the flow rate stabilizes to 45-50L / (m 2 h); adjust the pressure to 0.10-0.15 MPa, and linearly increase the pressure to 0.30-0.35 MPa at a rate of 0.05 MPa / min; maintain the pressure at 0.30-0.35 MPa for 20-30 min, and then reduce the pressure to 0.05-0.20 MPa until no droplets are left; and obtain the filtrate; S4, ceramic membrane filtration: using a ceramic membrane to filter the filtrate to obtain a primary filtrate; S5. Ultrafiltration: Ultrafiltration is performed on the primary filtrate using a 30-50 kDa ultrafiltration membrane. When the volume of the retentate is concentrated to 1 / 2 of the original volume of the primary filtrate, the filtration mode is switched to constant volume diafiltration: physiological saline is added at a rate of 2.5-3.0 L / min, and the permeate is discharged at the same rate, displacing a cumulative volume of 3-5 times the original volume of the primary filtrate. When the conductivity is ≤1.5 mS / cm and UV280 is ≤0.02, the diafiltration is stopped and the retentate is further concentrated to 1 / 4-1 / 5 of the original volume of the primary filtrate. The retentate is finally pre-filtered through the membrane to obtain the ultrafiltrate. S6, UV inactivation: A medium-pressure mercury lamp array is installed in the cavity to pass the ultrafiltrate through the cavity, with a cumulative irradiation dose of 20-30 kJ / m 2 ; In the medium pressure mercury lamp array, 185nm accounts for 10-15% and 254nm accounts for 85-90%; S7. Subpackaging: The inactivated solution is subpacked into sterile containers through a sterile filter to obtain a polydeoxyribonucleotide inactivated solution.

2. The inactivation process according to claim 1, wherein The depth filter in step S1 is a 5-20 μm depth filter; or the filtration flow rate in step S1 is 50-100 L / h.

3. The inactivation process according to claim 1, wherein The stirring in step S2 is stirring at 100-200 rpm for 10-30 min.

4. The inactivation process according to claim 1, wherein The material of the filter cloth in step S3 includes: any one or more of polypropylene, polyester fiber, polyamide, polyvinyl formal fiber, and polytetrafluoroethylene.

5. The inactivation process according to claim 1, characterized in that The pore size of the filter cloth in step S3 is 5-10 μm.

6. The inactivation process according to claim 1, characterized in that The pore size of the ceramic membrane in step S4 is 100-200 nm; or the membrane flux of the ceramic membrane filtration in step S4 is 100-120 LMH.

7. The inactivation process according to claim 1, characterized in that The inlet pressure of the ceramic membrane filtration in step S4 is 0.16-0.20 MPa; the outlet pressure is 0.08-0.10 MPa.

8. The inactivation process according to claim 1, characterized in that The inlet pressure of the ultrafiltration in step S5 is 0.1-0.2 MPa, and the outlet pressure is 0.05-0.1 MPa.

9. The inactivation process according to claim 1, characterized in that The membrane flux of the ultrafiltration in step S5 is 50-75LMH.

10. Use of the inactivation process according to any one of claims 1 to 9 in inactivating polydeoxyribonucleotides.

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