A method for detecting molecular weight distribution of pdrn / pn

Abstract

The application relates to the technical field of macromolecule analysis and detection, and discloses a PDRN / PN molecular weight distribution detection method, which comprises the following steps: S1, dissolving PDRN / PN samples in ultrapure water to prepare an aqueous phase mother liquor; S2, preparing three groups of to-be-detected samples for 50 mmol / L, 150 mmol / L and 300 mmol / L NaCl condition detection from the same aqueous phase mother liquor, wherein each group comprises at least three independently prepared to-be-detected samples; the PDRN / PN concentration in the three groups of to-be-detected samples is the same, and the NaCl concentration, buffer system concentration and pH of the to-be-detected samples in each group are consistent with corresponding running mobile phases; the three kinds of running mobile phases are the same in composition except for the NaCl concentration.

Description

Technical Field

[0001] This invention relates to the field of macromolecular analysis and detection technology, specifically a method for detecting the molecular weight distribution of PDRN / PN. Background Technology

[0002] PDRN (polydeoxyribonucleic acid) and PN (polynucleotide) are typically composed of deoxyribonucleic acid fragments of varying chain lengths, belonging to nucleic acid systems with a wide molecular weight distribution. For this type of sample, a single average molecular weight is insufficient to fully reflect its composition; molecular weight distribution parameters such as Mn, Mw, Mz, and PDI are usually required.

[0003] Gel electrophoresis can observe the migration range of nucleic acid fragments, but it is mainly used for qualitative or semi-quantitative determination. For mixtures of high-molecular-weight nucleic acids with wide distribution, mass spectrometry analysis is also easily limited by signal dispersion and the ability to detect long-chain fragments. SEC-MALS-RI can calculate molecular weight and its distribution by combining light scattering signals and differential refractive index concentration signals after size exclusion separation, and is therefore suitable for molecular weight characterization of PDRN or PN.

[0004] Both PDRN and PN are charged biomolecules. When using aqueous SEC for detection, the ionic composition of the mobile phase affects the hydration state of the nucleic acid chains and the non-size exclusion effect between the sample and the stationary phase. Lower salt concentrations may result in tailing, peak broadening, or reduced recovery. Increasing the salt concentration can weaken some electrostatic effects, but further increases in salt concentration do not necessarily lead to further improvement in detection results.

[0005] MALS determination itself does not depend on the retention time-molecular weight calibration curve, but the molecular weight distribution obtained by SEC-MALS-RI is still affected by factors such as whether the sample is completely eluted, the RI concentration signal, and the dn / dc setting. If different salt conditions cause selective retention, selective loss, changes in concentration signal, or other non-ideal chromatographic behaviors, the reported Mw may still change even if the sample main peak can still be integrated. These factors can exist individually or act in combination. Therefore, this method uses comparative results under controlled conditions to confirm the formal determination conditions, rather than using a single mechanism of action as a prerequisite for judgment.

[0006] Existing screening methods typically begin by determining usable mobile phases based on indicators such as peak shape, baseline, recovery, and repeatability. However, for PDRN or PN, simply determining whether the main peak under higher salt concentrations is integrable may not be sufficient to confirm whether the conditions are suitable for formal molecular weight determination. Therefore, it is necessary to compare Mw obtained at different NaCl concentrations, while ensuring comparability in sample source, sample concentration, buffer system, chromatographic conditions, and data processing conditions. Summary of the Invention

[0007] This invention provides a method for detecting the molecular weight distribution of PDRN / PN, which has the advantage of more accurate measurement results and solves the problem mentioned in the background art that existing methods tend to treat "the chromatographic peak can be integrated normally" and "the conditions are suitable for formal molecular weight determination" as the same judgment.

[0008] This invention provides the following technical solution: a method for detecting the molecular weight distribution of PDRN / PN, comprising the following steps: S1. Dissolve the PDRN / PN sample in ultrapure water to prepare an aqueous mother liquor; S2. Three sets of test samples are prepared from the same aqueous mother liquor for detection under conditions of 50 mmol / L, 150 mmol / L, and 300 mmol / L NaCl. Each set includes at least three independently prepared test samples. The PDRN / PN concentrations in the three sets of test samples are the same, and the NaCl concentration, buffer system concentration, and pH of each set of test samples are consistent with the corresponding operating mobile phase. The three operating mobile phases are identical in composition except for the NaCl concentration. S3. Under the same size exclusion chromatography, MALS detection, RI detection and data processing conditions, the three groups of samples to be tested were detected respectively, and the average weight-average molecular weight Mw of each group was calculated and recorded as Mw50, Mw150 and Mw300 respectively; when the main peak obtained under the 300 mmol / L NaCl condition can still be completely integrated, and Mw50 is greater than Mw150, Mw150 is greater than Mw300, and the RSD of Mw under the 150 mmol / L NaCl condition is not higher than 2.0%, the 150 mmol / L NaCl condition is determined as the formal determination condition; S4. Perform SEC-MALS-RI analysis on the PDRN / PN test sample according to the formal test conditions to obtain the number-average molecular weight Mn, weight-average molecular weight Mw, Z-average molecular weight Mz, and polydispersity index PDI.

[0009] As an optional scheme of the PDRN / PN molecular weight distribution detection method described in this invention, the three operating mobile phases all contain a 50 mmol / L phosphate buffer system, and the pH is 7.0.

[0010] As an optional scheme of the PDRN / PN molecular weight distribution detection method of the present invention, in step S1, the PDRN / PN sample is dissolved in ultrapure water without ultrasonic disruption and high-speed shearing, and the PDRN / PN concentration of the resulting aqueous mother liquor is 1.0 mg / mL; the PDRN / PN concentration in the sample to be tested is 0.050 mg / mL.

[0011] As an optional scheme of the PDRN / PN molecular weight distribution detection method of the present invention, wherein: 0.50 mL of the aqueous mother liquor is measured and prepared to 10.00 mL with a sample conditioning solution that matches the corresponding operating mobile phase; the sample conditioning solution compensates for the volume of water brought in by the aqueous mother liquor, so that the NaCl concentration, phosphate buffer system concentration and pH of the sample to be tested are consistent with the corresponding operating mobile phase.

[0012] As an optional scheme of the PDRN / PN molecular weight distribution detection method of the present invention, wherein: when the main peak obtained under 300 mmol / L NaCl conditions can be completely integrated, the sample recovery rate is 85% to 95%, and the PDI is 1.3 to 1.6, the Mw300 under this condition is calculated, and the Mw300 is compared with Mw150 and Mw50, wherein Mw50, Mw150 and Mw300 satisfy Mw50 > Mw150 > Mw300.

[0013] As an optional embodiment of the PDRN / PN molecular weight distribution detection method described in this invention, wherein: before detecting the PDRN / PN test sample, salmon testis DNA sodium salt is used as a reference standard for system suitability testing and SEC-MALS-RI detection is performed, and the reference molecular weight of the salmon testis DNA sodium salt is approximately 1.3 × 10⁻⁶. 6 Da.

[0014] As an optional scheme of the PDRN / PN molecular weight distribution detection method described in this invention, the same data processing model and baseline determination principle are used under different NaCl concentration conditions; the refractive index increment dn / dc of PDRN / PN is 0.158 mL / g.

[0015] As an optional scheme of the PDRN / PN molecular weight distribution detection method of the present invention, wherein: the NaCl concentration conditions are all set to be the same as the blank with the same composition as the corresponding running mobile phase; the sample to be tested is filtered through a 0.22 μm low adsorption polyethersulfone aqueous phase filter membrane before being injected.

[0016] As an optional scheme of the PDRN / PN molecular weight distribution detection method of the present invention, the size exclusion chromatographic column is filled with a fully porous SEC bonded phase with a particle size of 2.7 μm, the mobile phase flow rate is 0.50 mL / min, and the single injection volume is 500 μL.

[0017] As an optional scheme of the PDRN / PN molecular weight distribution detection method of the present invention, if step S3 does not satisfy Mw50 greater than Mw150 and Mw150 greater than Mw300, or the RSD of Mw is higher than 2.0% under 150 mmol / L NaCl conditions, the system suitability test is first repeated, then the aqueous mother liquor and three sets of samples to be tested are prepared again, and steps S2 and S3 are repeated.

[0018] The present invention has the following beneficial effects: This invention overcomes the limitations of traditional methods that rely solely on the integrity of chromatographic peaks to determine the suitability of the mobile phase. Under 300 mmol / L NaCl conditions, where the main peak is still integrable, the invention further compares the trends of weight-average molecular weight (Mw) measured at salt concentrations of 50, 150, and 300 mmol / L. Only when Mw50 > Mw150 > Mw300 and the RSD ≤ 2.0% under 150 mmol / L conditions is the condition considered the official measurement condition. This avoids molecular weight measurement deviations caused by non-size exclusion effects under high-salt conditions, significantly improving the reliability of the measurement results.

[0019] This invention uses the same aqueous mother liquor to prepare three sets of comparative samples, and uses a pre-compensated sample conditioning solution to accurately compensate for the water volume brought in by the mother liquor, so that the ionic composition of the sample to be tested is completely consistent with the corresponding mobile phase. This effectively eliminates the influence of sample matrix differences on light scattering and differential signals, and ensures the fairness and accuracy of Mw comparison under different salt concentration conditions.

[0020] This invention clarifies a repeatability quantification index for Mw under 150 mmol / L NaCl conditions, with an RSD not exceeding 2.0%, ensuring the precision of the formal determination conditions. In the example, the RSD of three determinations was only 1.40%, indicating that the method has good repeatability and stability and is suitable for industrial quality control detection.

[0021] Under confirmed formal measurement conditions, this invention can simultaneously obtain complete molecular weight distribution parameters such as number-average molecular weight (Mn), weight-average molecular weight (Mw), Z-average molecular weight (Mz), and polydispersity index (PDI), overcoming the shortcomings of traditional gel electrophoresis, which can only make qualitative or semi-quantitative judgments. It provides an accurate and comprehensive quantitative means for the quality characterization of PDRN or PN-type broad-distribution nucleic acid samples. Attached Figure Description

[0022] Figure 1 This is a flowchart of the method of the present invention. Figure 2 This is a flowchart illustrating the determination of measurement conditions for the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0024] A method for detecting the molecular weight distribution of PDRN / PN includes the following steps: S1. Dissolve the PDRN / PN sample in ultrapure water to prepare an aqueous mother liquor; S2. Three sets of test samples were prepared from the same aqueous mother liquor for detection under NaCl conditions of 50 mmol / L, 150 mmol / L, and 300 mmol / L. Each set included at least three independently prepared test samples. The PDRN / PN concentrations in the three sets of test samples were the same, and the NaCl concentration, buffer system concentration, and pH of each set of test samples were consistent with the corresponding operating mobile phase. The three operating mobile phases were identical in composition except for the NaCl concentration. S3. Under the same size exclusion chromatography, MALS detection, RI detection and data processing conditions, three groups of samples were detected respectively. The average weight-average molecular weight Mw of each group was calculated and recorded as Mw50, Mw150 and Mw300 respectively. When the main peak obtained under the 300 mmol / L NaCl condition can still be completely integrated, and Mw50 is greater than Mw150 and Mw150 is greater than Mw300, and the RSD of Mw under the 150 mmol / L NaCl condition is not higher than 2.0%, the 150 mmol / L NaCl condition is determined as the formal determination condition. S4. Perform SEC-MALS-RI analysis on the PDRN / PN test sample according to the formal test conditions to obtain the number-average molecular weight Mn, weight-average molecular weight Mw, Z-average molecular weight Mz, and polydispersity index PDI.

[0025] All three operating mobile phases contained a 50 mmol / L phosphate buffer system, and the pH was 7.0.

[0026] In step S1, the PDRN / PN test sample is dissolved in ultrapure water without ultrasonic disruption and high-speed shearing, and the PDRN / PN concentration of the resulting aqueous mother liquor is 1.0 mg / mL; the PDRN / PN concentration in the sample to be tested is 0.050 mg / mL.

[0027] Take 0.50 mL of the aqueous mother liquor and prepare a sample conditioning solution that matches the corresponding operating mobile phase to 10.00 mL. The sample conditioning solution compensates for the water volume brought in by the aqueous mother liquor, so that the NaCl concentration, phosphate buffer system concentration and pH of the sample to be tested are consistent with the corresponding operating mobile phase.

[0028] When the main peak obtained under 300 mmol / L NaCl conditions can be fully integrated, the sample recovery rate is 85%–95%, and the PDI is 1.3–1.6, calculate Mw300 under these conditions, and compare Mw300 with Mw150 and Mw50, wherein Mw50, Mw150, and Mw300 satisfy Mw50 > Mw150 > Mw300.

[0029] Before testing the PDRN / PN sample, salmon testicular DNA sodium salt was used as a control for system suitability testing and analyzed by SEC-MALS-RI. The reference molecular weight of salmon testicular DNA sodium salt is approximately 1.3 × 10⁻⁶. 6 Da.

[0030] The same data processing model and baseline determination principle were used under different NaCl concentration conditions; the refractive index increment dn / dc of PDRN / PN was 0.158 mL / g, or the dn / dc measured under the corresponding operating mobile phase conditions and verified by the method was used.

[0031] All NaCl concentration conditions were set to be blanks with the same composition as the corresponding operating mobile phase; the samples to be tested were filtered through a 0.22 μm low-adsorption polyethersulfone aqueous phase filter membrane before being injected.

[0032] The size exclusion column was packed with a fully porous SEC bonded phase with a particle size of 2.7 μm, the mobile phase flow rate was 0.50 mL / min, and the single injection volume was 500 μL.

[0033] If step S3 does not satisfy the condition that Mw50 is greater than Mw150 and Mw150 is greater than Mw300, or the RSD of Mw is higher than 2.0% under 150 mmol / L NaCl conditions, the system suitability test should be repeated first, then the aqueous mother liquor and three sets of test samples should be prepared again, and steps S2 and S3 should be repeated.

[0034] This invention uses the same aqueous mother liquor as a starting point for comparison. Multiple samples to be tested were prepared independently under NaCl conditions of 50 mmol / L, 150 mmol / L, and 300 mmol / L, while maintaining consistent PDRN or PN concentrations, buffer systems, pH, instrumentation, and data processing conditions. The ionic composition of each sample to be tested was matched with the corresponding operating flow to reduce the influence of differences in the injection matrix on the comparison results.

[0035] The average value of Mw was obtained under three NaCl conditions. When the main peak under the 300 mmol / L NaCl condition could still be fully integrated, the Mw obtained under the 50 mmol / L condition was higher than that under the 150 mmol / L condition, and the Mw obtained under the 300 mmol / L condition was lower than that under the 150 mmol / L condition, the repeatability of the 150 mmol / L condition was further investigated. When the RSD of Mw under the 150 mmol / L condition was not higher than 2.0%, this condition was used as the official determination condition.

[0036] Mn, Mw, Mz, and PDI were formally determined using SEC-MALS-RI. A 50 mmol / L phosphate buffer system at pH 7.0 was preferred, and the mobile phase composition, except for the NaCl concentration, remained constant throughout the comparison process.

[0037] Example 1, please refer to and Figure 2 Prior to formal testing, sodium salt of salmon testis DNA was used as a control for system suitability testing and analyzed using SEC-MALS-RI. The data for the materials used specified a Na content of no less than 5.0%, with a batch result of 6.5%; an A260 specification of no less than 15, with a batch result of 17; and a reference molecular weight of approximately 1.3 × 10⁻⁶. 6 Da. This reference molecular weight is used to compare the system's operating status and is not used as the basis for assigning values ​​to the PDRN or PN to be measured.

[0038] The system suitability test must be performed at least three times consecutively. During the test, it must be confirmed that the main peak can be stably integrated, the RI and MALS signals correspond normally, the baseline can be recovered, and the repeated measurements meet the pre-established system control requirements. If the system suitability test does not meet the requirements, subsequent NaCl condition comparisons will not be performed.

[0039] When it is necessary to validate the system response over a wider molecular weight range, DNA references at different chain length levels can be added. For example, two or more DNA references of 100 bp, 1500 bp, 5000 bp, and 13000 bp can be selected for detection range validation. This validation is used to supplement system response information at different molecular weight levels and does not replace routine system suitability testing of salmon testis DNA sodium salts.

[0040] Example 2 Preparation of aqueous mother liquor and operating mobile phase: Accurately weigh 50.0 mg of PDRN or PN sample and place it in a 50.0 mL volumetric flask. Add approximately 40 mL of ultrapure water. Dissolve the sample while avoiding ultrasonic disruption and high-speed shearing. After complete dissolution, dilute to the mark with ultrapure water and mix well to obtain a 1.0 mg / mL aqueous stock solution.

[0041] Three operating mobile phases with NaCl concentrations of 50 mmol / L, 150 mmol / L, and 300 mmol / L were prepared. Each mobile phase contained a 50 mmol / L phosphate buffer system, and the pH was adjusted to 7.0. The mobile phases were degassed after filtration through a 0.22 μm aqueous filter membrane; in one embodiment, ultrasonic degassed for 15 min.

[0042] Example 3 For each NaCl condition, at least three samples were prepared independently from the same aqueous mother liquor. For each sample, 0.50 mL of the aqueous mother liquor was accurately measured and diluted to 10.00 mL with the corresponding sample conditioning solution. The resulting PDRN or PN concentration was 0.050 mg / mL.

[0043] The solvent for the aqueous mother liquor is ultrapure water. If the running mobile phase is used directly for dilution, the water introduced by the mother liquor will cause the salt and buffer components in the final sample to be lower than those in the running mobile phase. Therefore, a pre-compensated concentration is used in the sample conditioning solution to ensure that the concentrations of NaCl and phosphate buffer system after mixing are consistent with the corresponding running mobile phase.

[0044] Table 1: Comparison of sample preparation conditions and results 50 mmol / L NaCl 52.63 mmol / L 52.63 mmol / L 0.50 mL / 9.50 mL PDRN or PN 0.050 mg / mL; NaCl 50 mmol / L; Phosphate 50 mmol / L 150 mmol / L NaCl 157.89 mmol / L 52.63 mmol / L 0.50 mL / 9.50 mL PDRN or PN 0.050 mg / mL; NaCl 150 mmol / L; Phosphate 50 mmol / L 300 mmol / L NaCl 315.79 mmol / L 52.63 mmol / L 0.50 mL / 9.50 mL PDRN or PN 0.050 mg / mL; NaCl 300 mmol / L; Phosphate 50 mmol / L The sample conditioning solution was adjusted to pH 7.0 using a phosphate system. After mixing with the aqueous mother liquor, the final sample pH was measured and controlled within 7.0 ± 0.05. If deviations were found, the solution was adjusted to this range with a small amount of phosphoric acid or sodium hydroxide solution before final volume determination, and then the corresponding sample conditioning solution was used for final volume determination. When using other sampling volumes or final volumes, the concentration of the sample conditioning solution could be adjusted according to the same material balance principle.

[0045] Example 4 Chromatographic, detection, and data processing conditions: Size-limited columns suitable for biomacromolecule analysis were used. The columns were packed with a fully porous SEC-bonded phase with a particle size of 2.7 μm. The mobile phase flow rate was 0.50 mL / min, and the single injection volume was 500 μL. The same column was used for all three NaCl conditions, and the column temperature, flow rate, injection volume, MALS detection settings, and RI detection settings were kept consistent.

[0046] The sample to be tested was filtered through a 0.22 μm low-adsorption polyethersulfone aqueous filter membrane before injection. During method validation, the sample recoveries and molecular weight distributions before and after filtration were compared to confirm that the filter membrane did not cause significant selective loss of specific molecular weight components. Blanks with the same composition as the operating mobile phase were set for each NaCl condition to identify non-sample signals generated by salt, injection, or baseline.

[0047] Mw calculations use RI concentration signals and MALS scattering signals. For DNA-based PDRN or PN, dn / dc is set to 0.158 mL / g in one implementation. The same data processing model and baseline determination principles are used for different NaCl conditions. Alternatively, dn / dc can be measured separately under the corresponding mobile phase conditions; if a fixed dn / dc is used, method validation should be performed to confirm that this setting does not change the comparative results.

[0048] The term "the main peak can be fully integrated" as used in this specification means that the main sample peaks corresponding to the RI and MALS signals are continuous and the integration start and end points can be determined according to a unified baseline determination principle. If the main peak is severely split, the baseline continues to drift, or integration cannot be completed according to the unified principle, then it does not qualify as "the main peak can be fully integrated".

[0049] Example 5 Comparison of different NaCl concentrations: At least three samples were prepared independently under NaCl conditions of 50 mmol / L, 150 mmol / L, and 300 mmol / L. The mean Mw and RSD of each group were calculated, and the mean Mw of the three groups were denoted as Mw50, Mw150, and Mw300.

[0050] If the main peak under the 300 mmol / L NaCl condition is severely split, the baseline cannot be recovered, or integration cannot be completed, then this condition will no longer be considered as a candidate for formal determination. If the main peak can still be fully integrated, then continue comparing Mw300 and Mw150, and further compare Mw50 and Mw150.

[0051] When Mw50 is greater than Mw150 and Mw150 is greater than Mw300, and the RSD of Mw under the 150 mmol / L NaCl condition is not higher than 2.0%, the 150 mmol / L NaCl condition is determined as the formal determination condition. This judgment is used to confirm the relative change of Mw reported under the three NaCl conditions, and does not indicate that PDRN or PN molecules will necessarily undergo chemical degradation or chain breakage under high salt conditions.

[0052] In a further defined embodiment, the sample recovery rate under 300 mmol / L NaCl conditions is 85%–95%, and the PDI is 1.3–1.6. Even if the above indicators do not show obvious abnormalities, as long as Mw300 is lower than Mw150 and Mw50 is higher than Mw150, the 300 mmol / L NaCl condition will not be used as the formal determination condition.

[0053] Example 6 Table 2: Comparison of NaCl development conditions 0 mmol / L Severe tailing or forward extension, peak dispersion <70% Mw is too high, Mn is too low, and PDI > 2.0 >10% Low-salinity boundary conditions 50 mmol / L Improved trailing effect, but may still result in asymmetry. 75%~85% When Mw is above 150 mmol / L, PDI is 1.5–2.0. 5%~8% Low salt comparison conditions 150mmol / L The main peak is symmetric and can be repeatedly integrated. 90%~110% Mw and Mn are stable, PDI is 1.2~1.5 <3% Formal determination of candidate conditions 300mmol / L The main peak can be fully integrated, and may extend forward. 85%~95% When Mw is below 150 mmol / L, the PDI is 1.3–1.6. 3%~5% Higher salt comparison conditions 500mmol / L Increased forward extension leads to decreased resolution. 80%~90% Mw further decreases, PDI stability declines. >5% High salinity boundary conditions 0 mmol / L and 500 mmol / L were used to observe abnormalities that were easily identifiable under both extreme conditions. 50 mmol / L and 300 mmol / L were within the boundaries of both extremes and could still be effectively integrated and compared with Mw. The 150 mmol / L condition had both formal detection data and repeatability data, and therefore served as an intermediate candidate condition among the three comparison conditions.

[0054] Table 2 shows the variation of Mw in the reported results of the detection system under different salt conditions. This variation may be related to non-size exclusion, selective retention or loss, RI concentration signal, and dn / dc adaptation. This method does not consider any single factor as a necessary mechanism. By keeping other conditions consistent, NaCl concentration can be used as the primary comparison variable.

[0055] Example 7 The same batch of samples was tested three times independently using a 150 mmol / L NaCl, 50 mmol / L phosphate buffer system and pH 7.0. Table 3: Actual repeatability results under 150 mmol / L NaCl conditions 1st time 1,613,290 1,997,700 2,431,590 1.238 1,960,630 2nd time 1,510,710 1,943,030 2,402,180 1.286 2,010,250 3rd 1,458,330 1,976,670 2,493,270 1.355 2,103,530 average value 1,527,443 1,972,467 2,442,347 1.293 2,024,803 RSD 5.16% 1.40% 1.90% 4.55% 3.58% Example 8 Different PDRN or PN samples were tested according to the formal determination conditions of 150 mmol / L NaCl, 50 mmol / L phosphate buffer system, and pH 7.0. Table 4: Actual detection results of different PDRN or PN samples Sample A 1,375,670 1,874,150 2,371,570 1.362 2,001,940 Sample B 408,123 522,089 656,735 1.279 520,212 PN sample C 1,053,300 1,514,670 1,893,310 1.438 1,791,420 PDRN sample D 640,719 777,322 914,415 1.213 863,713 As shown in the table above, under the formal measurement conditions, molecular weight distribution parameters such as Mn, Mw, Mz, and PDI can be obtained for PDRN or PN samples at different molecular weight levels. System suitability testing is used to confirm the instrument and coupled system status; for a wider range of analyte molecular weights, the range can be verified by adding DNA references of different strand lengths.

[0056] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0057] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the molecular weight distribution of PDRN / PN, characterized in that: Includes the following steps: S1. Dissolve the PDRN / PN sample in ultrapure water to prepare an aqueous mother liquor; S2. Three sets of test samples are prepared from the same aqueous mother liquor for detection under NaCl conditions of 50 mmol / L, 150 mmol / L, and 300 mmol / L, respectively. Each set includes at least three independently prepared test samples. The PDRN / PN concentrations in the three sets of test samples are the same, and the NaCl concentration, buffer system concentration, and pH of each set of test samples are consistent with the corresponding operating mobile phase. The three operating mobile phases are identical in composition except for the NaCl concentration. S3. Under the same size exclusion chromatography, MALS detection, RI detection and data processing conditions, the three groups of samples to be tested were detected respectively, and the average weight-average molecular weight Mw of each group was calculated and recorded as Mw50, Mw150 and Mw300 respectively; when the main peak obtained under the 300 mmol / L NaCl condition can still be completely integrated, and Mw50 is greater than Mw150, Mw150 is greater than Mw300, and the RSD of Mw under the 150 mmol / L NaCl condition is not higher than 2.0%, the 150 mmol / L NaCl condition is determined as the formal determination condition; S4. Perform SEC-MALS-RI analysis on the PDRN / PN test sample according to the formal test conditions to obtain the number-average molecular weight Mn, weight-average molecular weight Mw, Z-average molecular weight Mz, and polydispersity index PDI.

2. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: All three operating mobile phases contained a 50 mmol / L phosphate buffer system, and the pH was 7.

0.

3. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: In step S1, the PDRN / PN test sample is dissolved in ultrapure water without ultrasonic disruption and high-speed shearing, and the PDRN / PN concentration of the resulting aqueous mother liquor is 1.0 mg / mL; the PDRN / PN concentration in the sample to be tested is 0.050 mg / mL.

4. The method for detecting the molecular weight distribution of PDRN / PN according to claim 3, characterized in that: Take 0.50 mL of the aqueous mother liquor and prepare it to 10.00 mL with a sample conditioning solution that matches the corresponding operating mobile phase. The sample conditioning solution compensates for the volume of water brought in by the aqueous mother liquor, so that the NaCl concentration, phosphate buffer system concentration and pH of the sample to be tested are consistent with the corresponding operating mobile phase.

5. The method for detecting the molecular weight distribution of PDRN / PN according to claim 4, characterized in that: When the main peak obtained under 300 mmol / L NaCl conditions can be fully integrated, the sample recovery rate is 85%–95%, and the PDI is 1.3–1.6, calculate Mw300 under these conditions, and compare Mw300 with Mw150 and Mw50, wherein Mw50, Mw150, and Mw300 satisfy Mw50 > Mw150 > Mw300.

6. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: Before testing the PDRN / PN sample, salmon testicular DNA sodium salt was used as a control for system suitability testing and analyzed by SEC-MALS-RI. The reference molecular weight of the salmon testicular DNA sodium salt is approximately 1.3 × 10⁻⁶. 6 Da.

7. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: The same data processing model and baseline determination principle were used under different NaCl concentration conditions; the refractive index increment dn / dc of PDRN / PN was 0.158 mL / g.

8. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: Each NaCl concentration condition was set to a blank with the same composition as the corresponding operating mobile phase; the sample to be tested was filtered through a 0.22 μm low-adsorption polyethersulfone aqueous phase filter membrane before being injected.

9. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: The size exclusion column is packed with a fully porous SEC bonded phase with a particle size of 2.7 μm, the mobile phase flow rate is 0.50 mL / min, and the single injection volume is 500 μL.

10. The method for detecting the molecular weight distribution of PDRN / PN according to claim 1, characterized in that: If step S3 does not satisfy the condition that Mw50 is greater than Mw150 and Mw150 is greater than Mw300, or the RSD of Mw is higher than 2.0% under 150 mmol / L NaCl conditions, the system suitability test should be repeated first, then the aqueous mother liquor and three sets of test samples should be prepared again, and steps S2 and S3 should be repeated.

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