A process for inactivating polydeoxyribonucleotides and uses thereof

Abstract

The present application belongs to the technical field of inactivation process, and particularly relates to a kind of polydeoxyribonucleotide inactivation process and application.The inactivation process of the present application is through deep filtration pretreatment, specific proportion of pickling diatomite and nanocellulose filter aid are dispersed under specific dissolved oxygen conditions, gradient pressure plate and frame filter, ceramic membrane filtration, ultrafiltration combined with constant volume infiltration, and specific wavelength proportion and dose UV inactivation, finally sterile dispensing obtains PDRN inactivated liquid.The PDRN inactivated liquid prepared by the inactivation process of the present application has DNA content of more than 99%, protein content of less than 0.2%, and endotoxin content of less than 0.005 EU / mL, while achieving efficient microbial inactivation, maximally retaining PDRN active ingredients, significantly improving product purity and safety, and having broad application prospects in the fields of medicine, medical aesthetics and biomedical science.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of inactivation processes, and particularly relates to a polydeoxyribonucleotide inactivation process and application. BACKGROUND

[0002] As a functional DNA fragment, polydeoxyribonucleotide (PDRN) 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-inflammatory and improving tissue microenvironment.

[0003] However, the industrialized preparation process of PDRN still faces multiple technical bottlenecks. On the one hand, the extraction of PDRN raw materials and subsequent processing steps are prone to introduce microbial contamination such as bacteria and viruses. If the inactivation process has defects, 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 strong chemical reagent treatment can easily cause DNA chain breakage, base oxidation or irreversible combination with impurities, resulting in the loss of active ingredients and significantly reducing product efficacy.

[0004] In the prior art, a single inactivation strategy cannot break through the balance dilemma between safety and activity. For example, high-pressure steam sterilization can efficiently kill microorganisms, but high temperature can cause denaturation of PDRN molecular structure. Low-intensity ultraviolet (UV) irradiation can reduce damage to active ingredients, but it is difficult to completely inactivate light-resistant microorganisms. Traditional filtration processes often cause non-specific adsorption of PDRN on the surface of filter materials or lead to the residual of impurities such as proteins and endotoxins due to improper selection of filter materials or rough control of parameters, thereby causing poor process stability, significant batch-to-batch quality fluctuations and other problems. In addition, there is a lack of systematic and collaborative optimization among process steps, which cannot form an integrated solution with efficient sterilization, active protection and precise removal of impurities.

[0005] Therefore, how to develop a collaborative 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 fields of biological medicine and inactivation process technology. SUMMARY

[0006] In view of the above problems, the application provides a polydeoxyribonucleotide inactivation process and application.

[0007] The technical scheme of the application is as follows:

[0008] In one aspect, the present application provides a process for inactivating polydeoxyribonucleotides, comprising the following steps:

[0009] S1, pretreatment: filtering the polydeoxyribonucleotide solution through a depth filter, and collecting the filtered liquid as a crude filtrate;

[0010] S2, filter aid dispersion: adding 0.50-0.55% w / v acid-washed diatomite and 0.40-0.45% w / v nanocellulose to the crude filtrate, and stirring to disperse them uniformly under the condition of 0.3-0.5 ppm dissolved oxygen;

[0011] S3, pressure filtration: using a plate-and-frame filter press for pressure filtration, pre-coating the filter cloth with an acid-washed diatomite suspension, and pressure filtering at 0.05-0.08 MPa until the flow rate stabilizes to 45-50 L / (m 2 ·h); adjusting the pressure to 0.10-0.15 MPa, linearly increasing the pressure to 0.30-0.35 MPa at a rate of 0.05 MPa / min; maintaining the pressure at 0.30-0.35 MPa for 20-30 min, then decreasing the pressure to 0.05-0.20 MPa until no liquid droplets are present; obtaining a pressure filtrate;

[0012] S4, ceramic membrane filtration: filtering the pressure filtrate using a ceramic membrane to obtain a primary filtrate;

[0013] S5, ultrafiltration: ultrafiltering 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, switching to a constant-volume diafiltration mode: supplementing physiological saline at a rate of 2.5-3.0 L / min while discharging the permeate at the same rate, and cumulatively replacing 3-5 times the original volume of the primary filtrate; when the conductivity is ≤1.5 mS / cm and the UV280 is ≤0.02, stopping the diafiltration and continuing to concentrate the retentate to 1 / 4-1 / 5 of the original volume of the primary filtrate; finally, obtaining an ultrafiltrate by pre-filtering the retentate through a filter membrane;

[0014] S6, UV inactivation: arranging a medium-pressure mercury lamp array in a cavity to make the ultrafiltrate pass through the cavity, and cumulatively irradiating the ultrafiltrate to a dose of 20-30 kJ / m 2 ; the medium-pressure mercury lamp array has a 185 nm proportion of 10-15% and a 254 nm proportion of 85-90%;

[0015] S7, dispensing: dispensing the inactivated solution into sterile containers through a sterile filter to obtain a polydeoxyribonucleotide inactivated solution.

[0016] Specifically, the depth filter in step S1 is a 5-20 μm depth filter.

[0017] Preferably, the depth filter in step S1 is a 10 μm depth filter.

[0018] Specifically, the flow rate of the filter described in step S1 is 50-100 L / h.

[0019] Preferably, the flow rate of the filter in step S1 is 70-75 L / h.

[0020] Specifically, the amount of 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.

[0021] Preferably, the amount of acid-washed diatomaceous earth added in step S2 is 0.50% w / v or 0.55% w / v.

[0022] 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.

[0023] Preferably, the amount of acid-washed diatomaceous earth added in step S2 is 0.40% w / v or 0.50% w / v.

[0024] Specifically, the material of the filter cloth mentioned in step S3 includes any one or more of the following: polypropylene, polyester fiber, polyamide, polyvinyl alcohol formaldehyde fiber, and polytetrafluoroethylene.

[0025] Preferably, the filter cloth described in step S3 is made of polypropylene.

[0026] Specifically, the pore size of the filter cloth mentioned in step S3 is 5-10 μm.

[0027] Preferably, the filter cloth described in step S3 has a pore size of 5 μm.

[0028] Specifically, the pore size of the ceramic membrane described in step S4 is 100-200 nm.

[0029] Preferably, the pore size of the ceramic membrane in step S4 is 100 nm or 200 nm.

[0030] Specifically, the membrane flux of the ceramic membrane filtration in step S4 is 100-120 LMH.

[0031] Preferably, the membrane flux of the ceramic membrane filtration in step S4 is 100-110 LMH or 110-120 LMH.

[0032] More preferably, the membrane flux of the ceramic membrane filtration in step S4 is 100 LMH or 120 LMH.

[0033] Specifically, the inlet pressure of the ceramic membrane filtration in step S4 is 0.16-0.20 Mpa; and the outlet pressure is 0.08-0.10 Mpa.

[0034] 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.

[0035] Preferably, the outlet pressure of the ceramic membrane filtration in step S4 is 0.08-0.09 Mpa or 0.09-0.10 Mpa.

[0036] 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.

[0037] Preferably, the inlet pressure of the ultrafiltration in step S5 is 0.2 Mpa, and the outlet pressure is 0.1 Mpa.

[0038] Specifically, the filter membrane in step S5 is a 0.45-1.0 μm filter membrane.

[0039] Preferably, the filter membrane in step S5 is a 0.45 μm filter membrane.

[0040] Specifically, the sterile filter in step S7 is a 0.1-0.22 μm sterile filter.

[0041] Preferably, the sterile filter in step S7 is a 0.22 μm sterile filter.

[0042] In another aspect, the present application provides the use of the inactivation process as described in any one of the above aspects for inactivating polydeoxyribonucleotides.

[0043] The present application has the following advantages:

[0044] The PDRN inactivation solution prepared by the inactivation process of the present application has a DNA content of more than 99%, a protein content of less than 0.2%, and an endotoxin content of less than 0.005 EU / mL, which realizes efficient microbial inactivation while maximizing the retention of active ingredients of PDRN, significantly improves the purity and safety of the product, and has a broad application prospect in the fields of medicine, medical aesthetics and biomedical science. DETAILED DESCRIPTION

[0045] The application will be further clarified by the following examples. The examples are only a part of the application and are not used to limit the application. The experimental methods used in the following examples are conventional experiments. The materials and reagents used in the following examples are commercially available unless otherwise specified. The polydeoxyribonucleotides used in the application are produced by our company Hengyu Biopharm (Shandong) Co., Ltd.

[0046] Example 1

[0047] Accurately weigh PDRN and place it in a beaker. Add normal saline to it, heat in a 40°C water bath and stir until the PDRN dissolves to obtain a 10 mg / mL polydeoxyribonucleotide solution.

[0048] 1. Pretreatment: The polydeoxyribonucleotide solution is passed through a 10 μm depth filter, the pH is controlled at 7.0-7.5, the temperature is controlled at 40°C, the flow rate is controlled at 75 L / h, and the filtered liquid is collected as the crude filtrate.

[0049] 2. Filter aid dispersion: Add 0.55% w / v acid-washed diatomite and 0.45% w / v nanocellulose to the crude filtrate, pass in CO2 to make the dissolved oxygen 0.3 ppm, and stir at 150 rpm for 20 min to make it uniformly dispersed.

[0050] 3. Pressure filtration: Use a plate and frame pressure filter (filter cloth material: polypropylene, pore size 5 μm) for pressure filtration, precoat the filter cloth with a 0.2% w / v acid-washed diatomite suspension to form a 1-2 mm thin layer, and pressure filter at 0.08 MPa to a stable flow rate of 50 L / (m 2 ·h); adjust the pressure to 0.15 MPa, linearly increase the pressure to 0.35 MPa at a rate of 0.05 MPa / min; maintain 0.35 MPa for 20 min, then reduce the pressure to 0.10 MPa until no liquid droplets are present, to obtain the pressure filtrate.

[0051] 4. Ceramic membrane filtration: Use a ceramic membrane with a pore size of 200 nm, control the inlet pressure at 0.16 MPa and the outlet pressure at 0.08 MPa, and the membrane flux is 100 LMH, filter the pressure filtrate to obtain the primary filtrate.

[0052] 5. Ultrafiltration: The 50 kDa ultrafiltration membrane was used, the inlet pressure was controlled at 0.2 MPa, the outlet pressure was 0.1 MPa, the circulating flow rate was adjusted to 5 m / s, and the membrane flux was 75 LMH. The first-stage filtrate was subjected to ultrafiltration. When the volume of the retentate was concentrated to 1 / 2 of the original volume of the first-stage filtrate, the constant volume diafiltration mode was switched to: 37°C physiological saline was supplemented at a rate of 2.5 L / min, while the permeate was discharged at the same rate. The cumulative displacement was 3 times the original volume of the first-stage filtrate. The permeate was sampled every 30 minutes for detection: when the conductivity was ≤1.5 mS / cm and the UV280 was ≤0.02, the diafiltration was stopped, the retentate was concentrated to 1 / 4 of the original volume of the first-stage filtrate, and the final retentate was pre-filtered through a 0.45 μm filter membrane to obtain the ultrafiltrate.

[0053] 6. UV inactivation: The ultrafiltrate was irradiated in the cavity with a medium-pressure mercury lamp array (185 nm accounted for 15%, 254 nm accounted for 85%) until the cumulative irradiation dose reached 30 kJ / m 2 .

[0054] 7. Sub-packing: The solution after inactivation was sub-packed into sterile containers through a sterile filter (0.22 μm) to obtain the PDRN inactivated solution.

[0055] Example 2

[0056] The PDRN was accurately weighed and placed in a beaker. Physiological saline was added, heated in a 40°C water bath, and stirred until the PDRN was dissolved to obtain a 10 mg / mL polydeoxyribonucleotide solution.

[0057] 1. Pretreatment: The polydeoxyribonucleotide solution was filtered through a 10 μm depth filter. The pH was controlled at 7.0-7.5, the temperature was controlled at 40°C, and the flow rate was controlled at 70 L / h. The filtered liquid was collected as the crude filtrate.

[0058] 2. Filter aid dispersion: 0.50% w / v acid-washed diatomite and 0.40% w / v nanocellulose were added to the crude filtrate. CO2 was introduced to make the dissolved oxygen 0.5 ppm, and stirred at 150 rpm for 20 min to make it uniformly dispersed.

[0059] 3. Pressure filtration: A plate-and-frame pressure filter (filter cloth material: polypropylene, pore size 5 μm) was used for pressure filtration. A 0.4% w / v acid-washed diatomite suspension was used to pre-coat the filter cloth to form a 1-2 mm thin layer. The pressure was 0.08 MPa, and the flow rate was stabilized to 45 L / (m 2 ·h); the pressure was adjusted to 0.10 MPa, and linearly increased to 0.30 MPa at a rate of 0.05 MPa / min; after maintaining at 0.30 MPa for 30 min, the pressure was reduced to 0.10 MPa until no liquid droplets were observed to obtain the pressure filtrate.

[0060] 4. Ceramic membrane filtration: The ceramic membrane with a pore size of 100 nm was used, the inlet pressure was controlled at 0.2 MPa, and the outlet pressure was controlled at 0.1 MPa, and the membrane flux was 120 LMH. The pressure filtrate was filtered to obtain a primary filtrate.

[0061] 5. Ultrafiltration: The ultrafiltration membrane with a molecular weight cut-off of 30 kDa was used, the inlet pressure was controlled at 0.2 MPa, and the outlet pressure was 0.1 MPa. The circulating flow rate was adjusted to 5 m / s, and the membrane flux was 50 LMH. The primary filtrate was subjected to ultrafiltration. When the volume of the retentate was concentrated to 1 / 2 of the original volume of the primary filtrate, the constant volume diafiltration mode was switched to: 37°C physiological saline was added at a rate of 3.0 L / min, and the permeate was discharged at the same rate. The cumulative displacement was 5 times the original volume of the primary filtrate. Every 30 minutes, the permeate was sampled and detected: when the conductivity was ≤1.5 mS / cm and the UV280 was ≤0.02, the diafiltration was stopped, and the retentate was concentrated to 1 / 5 of the original volume of the primary filtrate. The final retentate was pre-filtered through a 0.45 μm filter membrane to obtain an ultrafiltrate.

[0062] 6. UV inactivation: The ultrafiltrate was irradiated in a cavity equipped with a medium-pressure mercury lamp array (185 nm accounted for 10%, 254 nm accounted for 90%) until the cumulative irradiation dose reached 20 kJ / m 2 .

[0063] 7. Dispensing: After inactivation, the solution was dispensed into sterile containers through a sterile filter (0.22 μm) to obtain PDRN inactivated solution.

[0064] Comparative Example 1

[0065] The difference between Comparative Example 1 and Example 1 is only that the "2, filter aid dispersion" is different, and the specific steps are as follows:

[0066] 2. Filter aid dispersion: 0.45% w / v acid-washed diatomite and 0.55% w / v nanocellulose were added to the crude filtrate, CO2 was introduced to make the dissolved oxygen 0.7 ppm, and stirring was carried out at 150 rpm for 20 min to make it uniformly dispersed.

[0067] Comparative Example 2

[0068] The difference between Comparative Example 2 and Example 1 is only that the "2, filter aid dispersion" is different, and the specific steps are as follows:

[0069] 2. Filter aid dispersion: 0.60% w / v acid-washed diatomite and 0.40% w / v nanocellulose were added to the crude filtrate, CO2 was introduced to make the dissolved oxygen 0.2 ppm, and stirring was carried out at 150 rpm for 20 min to make it uniformly dispersed.

[0070] Comparative Example 3

[0071] The difference between Comparative Example 3 and Example 1 is only that the "3, pressure filtration" is different, and the specific steps are as follows:

[0072] 3. Pressure filtration: using plate-and-frame filter press (filter cloth material: polypropylene, pore size 5 pm) for pressure filtration, pre-coating the filter cloth with 0.2% w / v acid-washed diatomite suspension to form a 1-2 mm thin layer, pressure filtration at 0.05 MPa until the flow rate is stable at 50 L / (m2-h); adjusting the pressure to 0.10 MPa, linearly increasing the pressure to 0.50 MPa at a rate of 0.05 MPa / min; after maintaining at 0.50 MPa for 20 min, reducing the pressure to 0.10 MPa until no liquid droplets are present, to obtain the pressure filtrate. 2 • h); adjusting the pressure to 0.10 MPa, linearly increasing the pressure to 0.50 MPa at a rate of 0.05 MPa / min; after maintaining at 0.50 MPa for 20 min, reducing the pressure to 0.10 MPa until no liquid droplets are present, to obtain the pressure filtrate.

[0073] Comparative Example 4

[0074] Comparative Example 4 differs from Example 1 only in that “3. Pressure filtration” is different, and the specific steps are as follows:

[0075] 3. Pressure filtration: using plate-and-frame filter press (filter cloth material: polypropylene, pore size 5 pm) for pressure filtration, pre-coating the filter cloth with 0.2% w / v acid-washed diatomite suspension to form a 1-2 mm thin layer, pressure filtration at 0.08 MPa until the flow rate is stable at 40 L / (m2-h); adjusting the pressure to 0.20 MPa, linearly increasing the pressure to 0.35 MPa at a rate of 0.05 MPa / min; after maintaining at 0.35 MPa for 40 min, reducing the pressure to 0.10 MPa until no liquid droplets are present, to obtain the pressure filtrate. 2 • h); adjusting the pressure to 0.10 MPa, linearly increasing the pressure to 0.50 MPa at a rate of 0.05 MPa / min; after maintaining at 0.50 MPa for 20 min, reducing the pressure to 0.10 MPa until no liquid droplets are present, to obtain the pressure filtrate.

[0076] Comparative Example 5

[0077] Comparative Example 5 differs from Example 1 only in that “5. Ultrafiltration” is different, and the specific steps are as follows:

[0078] 5. Ultrafiltration: using a 50 kDa ultrafiltration membrane, the inlet pressure is controlled at 0.2 MPa, the outlet pressure is 0.1 MPa, the circulating flow rate is adjusted to 5 m / s, and the membrane flux is 75 LMH; when the volume of the retentate is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to the constant volume diafiltration mode: add 37°C physiological saline at a rate of 2.0 L / min, while discharging the permeate at the same rate, and the cumulative displacement is 2 times the original volume of the primary filtrate. Sample detection every 30 minutes: when the conductivity is ≤1.5 mS / cm and UV280 is ≤0.02, stop diafiltration, continue to concentrate the retentate to 1 / 3 of the original volume of the primary filtrate, and finally the retentate is pre-filtered through a 0.45 pm filter membrane to obtain the ultrafiltrate.

[0079] Comparative Example 6

[0080] Comparative Example 6 differs from Example 1 only in that “5. Ultrafiltration” is different, and the specific steps are as follows:

[0081] 5. Ultrafiltration: The primary filtrate was subjected to ultrafiltration using a 50 kDa ultrafiltration membrane, with an inlet pressure controlled at 0.2 MPa, an outlet pressure of 0.1 MPa, and a circulating flow rate adjusted to 5 m / s, and a membrane flux of 75 LMH. When the volume of the retentate was concentrated to 1 / 2 of the original volume of the primary filtrate, the mode was switched to constant volume diafiltration: 37 °C physiological saline was supplemented at a rate of 4.0 L / min, while the permeate was discharged at the same rate, and the cumulative displacement was 8 times the original volume of the primary filtrate. The permeate was sampled and detected every 30 minutes: when the conductivity was ≤1.5 mS / cm and UV280 was ≤0.02, the diafiltration was stopped, the retentate was concentrated to 1 / 4 of the original volume of the primary filtrate, and the final retentate was pre-filtered through a 0.45 μm filter membrane to obtain the ultrafiltrate.

[0082] Comparative Example 7

[0083] Comparative Example 7 and Example 1 differ only in that "2, filter aid dispersion", "3, pressure filtration", and "5, ultrafiltration" are different, and the specific steps are as follows:

[0084] 2. Filter aid dispersion: 0.45% w / v acid-washed diatomite and 0.55% w / v nanocellulose were added to the crude filtrate, CO2 was introduced to make the dissolved oxygen 0.7 ppm, and stirring was performed at 150 rpm for 20 min to make the dispersion uniform.

[0085] 3. Pressure filtration: A plate-and-frame pressure filter (filter cloth material: polypropylene, pore size 5 μm) was used for pressure filtration, 0.2% w / v acid-washed diatomite suspension was used to pre-coat the filter cloth to form a 1-2 mm thin layer, and pressure filtration was performed at 0.08 MPa until the flow rate stabilized to 50 L / (m 2 ·h); the pressure was adjusted to 0.20 MPa, and linearly increased to 0.50 MPa at a rate of 0.05 MPa / min; after maintaining at 0.50 MPa for 20 min, the pressure was reduced to 0.10 MPa until no liquid droplets were present, and the pressure filtrate was obtained.

[0086] 5. Ultrafiltration: The primary filtrate was subjected to ultrafiltration using a 50 kDa ultrafiltration membrane, with an inlet pressure controlled at 0.2 MPa, an outlet pressure of 0.1 MPa, and a circulating flow rate adjusted to 5 m / s, and a membrane flux of 75 LMH. When the volume of the retentate was concentrated to 1 / 2 of the original volume of the primary filtrate, the mode was switched to constant volume diafiltration: 37 °C physiological saline was supplemented at a rate of 4.0 L / min, while the permeate was discharged at the same rate, and the cumulative displacement was 8 times the original volume of the primary filtrate. The permeate was sampled and detected every 30 minutes: when the conductivity was ≤1.5 mS / cm and UV280 was ≤0.02, the diafiltration was stopped, the retentate was concentrated to 1 / 4 of the original volume of the primary filtrate, and the final retentate was pre-filtered through a 0.45 μm filter membrane to obtain the ultrafiltrate.

[0087] Comparative Example 8

[0088] The difference between Comparative Example 8 and Example 1 is only that "6, UV inactivation" is different, and the specific steps are as follows:

[0089] 6, UV inactivation: a medium pressure mercury lamp array (185 nm accounts for 20%, 254 nm accounts for 80%) is arranged in the cavity to make the ultrafiltrate pass through the cavity, and the cumulative irradiation dose reaches 50 kJ / m 2 .

[0090] Comparative Example 9

[0091] The difference between Comparative Example 9 and Example 1 is only that "2, filter aid dispersion" and "6, UV inactivation" are different, and the specific steps are as follows:

[0092] 2, filter aid dispersion: 0.45% w / v acid-washed diatomite and 0.55% w / v nanocellulose are added to the crude filtrate, CO2 is introduced to make the dissolved oxygen 0.7 ppm, and 150 rpm stirring is carried out for 20 min to make it uniformly dispersed.

[0093] 6, UV inactivation: a medium pressure mercury lamp array (185 nm accounts for 20%, 254 nm accounts for 80%) is arranged in the cavity to make the ultrafiltrate pass through the cavity, and the cumulative irradiation dose reaches 50 kJ / m 2 .

[0094] Experimental Example 1: DNA content determination in PDRN inactivation solution

[0095] The PDRN inactivation solution of Example 1-Example 2 and Comparative Example 1-Comparative Example 9 is appropriately diluted with physiological saline so that the absorbance is within the linear range of the standard curve. The absorbance of the PDRN inactivation solution is measured at 260 nm with physiological saline as a blank. The DNA concentration in the PDRN inactivation solution is calculated according to the absorbance. The determination results are shown in Table 1:

[0096] Table 1: DNA content determination

[0097]

[0098] 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%. The adjustment of the single or composite process of Comparative Example 1-Comparative Example 9 compared with Example 1 all leads to a decrease in DNA content. The above results show that under the specific acid-washed diatomite and nanocellulose dosage, specific dissolved oxygen content, specific pressure filtration and ultrafiltration conditions, and specific UV inactivation conditions, the active ingredients of PDRN can be maximally preserved under the premise of microbial inactivation.

[0099] The only difference between Comparative Example 9 and Example 1 is the difference in "filter aid dispersion" and "UV inactivation". The only difference between Comparative Example 1 and Example 1 is the difference in "filter aid dispersion"; the only difference between Comparative Example 8 and Example 1 is the difference in "UV inactivation". Based on Comparative Example 9, the synergistic effect of the "filter aid dispersion" process and the "UV inactivation" process in the inactivation process of the present invention is evaluated.

[0100] Compared to Comparative Example 9, the DNA content in the PDRN inactivation solution of Comparative Example 1 increased by 3.78%, and the DNA content in the PDRN inactivation solution of Comparative Example 2 increased by 0.53%; the DNA content in Example 1 increased by 4.93%. It is evident that the inactivation process of this invention, combining the "filter aid dispersion" process and the "UV inactivation" process, has a synergistic effect of 1+1>2 (4.93%>3.78%+0.53%).

[0101] Experimental Example 2: Determination of Protein Content in PDRN Inactivation Solution

[0102] The PDRN inactivation solutions from Examples 1-2 and Comparative Examples 1-9 were appropriately diluted with physiological saline to ensure their absorbance was within the linear range of the standard curve. The protein content in the PDRN inactivation solutions was determined using a protein concentration assay kit (Sangon Biotech, C504031-1000). The results are shown in Table 2.

[0103] Table 2 Protein content determination

[0104]

[0105] The above results indicate that the protein content in the PDRN inactivation solution prepared by the inactivation process in Examples 1 and 2 is below 0.20%. This demonstrates that precisely controlling the inactivation process steps of this invention is key to reducing the protein content of the PDRN inactivation solution and improving product purity.

[0106] Experimental Example 3: Activity Determination of PDRN Inactivation Solution

[0107] 1. Adjust the HUVEC cell density to 1.25 × 10⁻⁶. 5 Count / ml; Add 200μL of 1.25×10⁻⁶ to a 96-well plate. 5 Cells per ml were incubated overnight at 37°C.

[0108] 2. Discard all cell supernatant, add 200 μL of EBM2 medium containing 0.5% FBS, and incubate at 37°C for 24 hours;

[0109] 3. Discard 100 μL of cell supernatant, create wounds on HUVEC cells using a scratcher, and wash twice with serum-free EBM2 medium.

[0110] 4. Place the cells in a live cell culture chamber and add 100 µg / mL of PDRN inactivation solution from Examples 1-2 and Comparative Examples 1-9, diluted with complete culture medium (EBM2 cell culture medium containing 0.5% FBS and 1% penicillin / streptomycin). Use the complete culture medium as a control group. Record images after 24 hours, analyze cell migration area using ImageJ, and calculate cell migration rate. The results are shown in Table 3.

[0111] Table 3 Cell migration rate determination

[0112]

[0113] The PDRN inactivation solution prepared by the inactivation process in Examples 1 and 2 exhibits cell migration-promoting biological activity. This further demonstrates that the inactivation process of the present invention, through multi-step synergistic optimization, maximizes the preservation of PDRN's biological activity while ensuring high purity (DNA > 99%, protein < 0.2%).

[0114] Experimental Example 4: Determination of Endotoxin Content in PDRN Inactivation Solution

[0115] Endotoxin was detected according to the photometric determination method in 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.

[0116] Table 4. Determination of Endotoxin Content

[0117]

[0118] The above results indicate that the endotoxin content in the PDRN inactivation solution of Examples 1-2 is less than 0.005 EU / mL, which meets the requirements.

[0119] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A process for inactivating polydeoxyribonucleotides, characterized in that, The inactivation process includes the following steps: S1. Pretreatment: The polydeoxyribonucleotide solution is filtered through a depth filter, and the filtered liquid is collected as 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, and stir to disperse it evenly under dissolved oxygen conditions of 0.3-0.5 ppm. S3. Filtration: Use a plate and frame filter press for filtration. Pre-coat the filter cloth with an acid-washed diatomaceous earth suspension. Filtrate at 0.05-0.08 MPa until the flow rate stabilizes at 45-50 L / (m³). 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 0.30-0.35 MPa for 20-30 min, then decrease the pressure to 0.05-0.20 MPa until no droplets remain; obtain the filtrate; S4. Ceramic membrane filtration: The filtrate is filtered using a ceramic membrane to obtain a primary filtrate. S5. Ultrafiltration: Use a 30-50kDa ultrafiltration membrane to ultrafilter the primary filtrate. When the volume of the retentate is concentrated to 1 / 2 of the original volume of the primary filtrate, switch to constant volume percolation mode: add physiological saline at a rate of 2.5-3.0L / min, and discharge the permeate at the same rate, cumulatively replacing 3-5 times the original volume of the primary filtrate. When the conductivity is ≤1.5mS / cm and UV280 is ≤0.02, stop percolation and continue to concentrate the retentate to 1 / 4-1 / 5 of the original volume of the primary filtrate. Finally, the retentate is pre-filtered through a filter membrane to obtain the ultrafiltrate. S6. UV Inactivation: A medium-pressure mercury lamp array is installed in the chamber to allow the ultrafiltrate to pass through, with a cumulative irradiation dose of 20-30 kJ / m³. 2 In the aforementioned medium-pressure mercury lamp array, 185nm accounts for 10-15% and 254nm accounts for 85-90%. S7. Dispensing: After inactivation, the solution is dispensed into sterile containers through a sterile filter to obtain polydeoxyribonucleic acid inactivation solution.

2. The inactivation process according to claim 1, characterized in that, The deep filter mentioned in step S1 is a 5-20μm deep filter; or the flow rate of the filter mentioned in step S1 is 50-100L / h.

3. The inactivation process according to claim 1, characterized in that, The stirring described in step S2 is stirring at 100-200 rpm for 10-30 minutes.

4. The inactivation process according to claim 1, characterized in that, The filter cloth material mentioned in step S3 includes any one or more of the following: polypropylene, polyester fiber, polyamide, polyvinyl alcohol formaldehyde fiber, and polytetrafluoroethylene.

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

6. The inactivation process according to claim 1, characterized in that, The pore size of the ceramic membrane described in step S4 is 100-200 nm; or the membrane flux of the ceramic membrane filtration described 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 filter described 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 device described 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 50-75 LMH.

10. The application of the inactivation process according to any one of claims 1-9 in the inactivation of polydeoxyribonucleotides.

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