Method for detecting purity of pegylated recombinant human erythropoietin
After de-N sugar treatment and SDS denaturation treatment, capillary gel electrophoresis technology was used to detect the purity of PEGylated recombinant human elutin, which solved the problem of insufficient accuracy of traditional methods and achieved higher detection accuracy and reliability.
Patent Information
- Application Number
- CN202411710468.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-07
AI Technical Summary
The traditional SDS-PAGE method has poor accuracy when detecting the purity of pegylated recombinant human erectin (PEG-EPO), and it is difficult to effectively separate PEG-EPO from uncoupled EPO in capillary electrophoresis technology.
By using PNGase F enzyme for deN sugar treatment, the high microscopic inhomogeneity of PEG-EPO was reduced, and then SDS was added for superheating denaturation, destroying the secondary and tertiary structures, and finally using capillary gel electrophoresis for separation detection.
It improves the accuracy and reliability of purity detection, can be more accurate in quantification, and is suitable for promotion and application.
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Abstract
Description
Technical Field
[0001] The invention relates to a purity detection method for PEGylated recombinant human erythropoietin. Background Art
[0002] Recombinant human erythropoietin (EPO) is an effective drug for the treatment of renal anemia. Pegylated recombinant human erythropoietin (PEG-EPO) is a long-acting erythropoietin developed by modifying EPO with polyethylene glycol. Its half-life in the blood circulation is significantly prolonged, which greatly reduces the frequency of injections and significantly improves the limitations of the drug in clinical practice.
[0003] Purity is a key quality attribute (CQA) of protein drugs and is closely related to the safety and efficacy of the product. In the pharmacopoeias of various countries and regions, continuous product purity testing and control are required throughout the life cycle of drug process development. The Chinese Pharmacopoeia recommends the use of size exclusion chromatography (SEC-HPLC), reverse phase chromatography (RP-HPLC) and SDS-PAGE methods to test the purity of EPO and PEGylated recombinant protein products. However, traditional SDS-PAGE plate electrophoresis technology can only meet semi-quantitative requirements when detecting size variants, and the accuracy is poor.
[0004] Capillary electrophoresis is widely used in purity testing of biological products because its separation performance, accuracy, precision, linearity and range, durability and other indicators are superior to SDS-PAGE. However, the microscopic heterogeneity of EPO is very high, and the modification of PEG-EPO chain further increases its structural complexity, resulting in the fact that although the molecular weight of PEG-EPO and unconjugated EPO is significantly different (60kD vs 30kD), they cannot be effectively separated by capillary electrophoresis. Summary of the invention
[0005] In order to solve the problems existing in the prior art, the present invention provides a method for detecting the purity of PEGylated recombinant human erythropoietin.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] The purpose of the present invention is to provide a method for detecting the purity of PEGylated recombinant human erythropoietin, comprising the following steps:
[0008] (1) using PNGase F enzyme to remove N-glycans from the PEGylated recombinant human erythropoietin to obtain PEG-EPO after N-glycan removal;
[0009] (2) adding SDS to denature the N-glycan-depleted PEG-EPO;
[0010] (3) Perform capillary gel electrophoresis and calculate the purity of the sample based on the peak area ratio of different components.
[0011] As a preferred embodiment, before step (1), the method further comprises pre-treating the PEGylated recombinant human erythropoietin to be tested to obtain pre-treated PEG-EPO; the pre-treatment is to concentrate the PEGylated recombinant human erythropoietin to be tested and replace the solution with an enzyme cleavage buffer.
[0012] As a preferred embodiment, the concentration is carried out using an ultrafiltration membrane; and / or
[0013] The enzyme digestion buffer was 40 mM PB at pH 7.5±0.1.
[0014] As a preferred embodiment, in step (1), the N-sugar removal treatment is to mix PNGase F enzyme and PEGylated recombinant human erythropoietin to be tested and centrifuge, and incubate the supernatant at 37±1°C for ≥16h.
[0015] As a preferred embodiment, in step (2), the PEG-EPO after N-glycan removal is mixed with the sample premix solution and then incubated for denaturation;
[0016] The sample premix solution is composed of the following components in volume: 9 parts of 2X Sample Buffer; 1 part of 250mM NEM;
[0017] The formula of the 2X Sample Buffer is: 12.56 mM disodium hydrogen phosphate, 27.44 mM sodium dihydrogen phosphate, 2% SDS, pH 6.5-9.0.
[0018] As a preferred embodiment, the pH value is 6.5.
[0019] As a preferred solution, after the N-glycosylated PEG-EPO is mixed with the sample premix solution, the final concentration of the N-glycosylated PEG-EPO is 1.0 mg / mL.
[0020] As a preferred embodiment, in step (2), the incubation denaturation conditions are: 70-90°C, incubation for 5-10 min.
[0021] As a preferred embodiment, the incubation denaturation conditions are: 70° C., incubation for 10 min.
[0022] As a preferred embodiment, in step (3), the collection method of capillary gel electrophoresis is: injection: 4600V, 20s; separation: 5750V, 30min.
[0023] Since EPO contains multiple glycosylation sites (3 N glycosylation sites, 1 O glycosylation site, and more than one hundred glycoforms have been identified), the microscopic heterogeneity is very high, which further leads to the inability to achieve effective separation by capillary electrophoresis. Therefore, the present invention uses N-glycosylation enzyme to reduce the high microscopic heterogeneity of PEG-EPO molecules, adds SDS for overheat denaturation treatment to destroy the secondary and tertiary structures, and then uses capillary electrophoresis technology to separate and detect samples according to molecular size, which solves the problem that the quantitative accuracy of the traditional SDS-PAGE method is poor. When the capillary electrophoresis technology is used to determine the purity of such drugs, the peak number is large, the peak shape is deformed, the main component and impurities cannot be effectively separated, and then the problem of being unable to be quantified is ensured, ensuring the accuracy and reliability of purity determination. The method of the present invention has the characteristics of higher specificity, more accurate quantification, and suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:
[0025] Figure 1 The electrophoretic patterns of PEG-EPO samples incubated with PNGase F for different time periods.
[0026] Figure 2 This is the electrophoresis spectrum of the PEG-EPO sample with 1X Sample buffer premix.
[0027] Figure 3 This is the electrophoresis spectrum of the PEG-EPO sample with 2X Sample buffer premix.
[0028] Figure 4 The electrophoresis patterns of PEG-EPO samples at different sample buffer pH values.
[0029] Figure 5 This is the electrophoresis spectrum of the PEG-EPO sample without 10kD internal standard.
[0030] Figure 6 The electrophoresis patterns of PEG-EPO samples under different denaturation conditions.
[0031] Figure 7 CE-SDS electrophoresis patterns of PEG-EPO and EPO samples for application of the method of the present invention.
[0032] Figure 8 Shown are the electrophoretic patterns of blank control solution, negative control solution and PEG-EPO DS sample solution.
[0033] Fig. 9This is the electrophoresis spectrum of the high temperature degradation sample of PEG-EPO DS.
[0034] Fig.10 It is the linear result between the initial concentration of the sample and the peak area of the main peak of the sample.
[0035] Fig.11 It is the linear result of the initial concentration of the sample and the total peak area of the sample.
[0036] Fig.12 Conventional CE-SDS electrophoresis patterns of PEG-EPO and EPO samples without N-glycoside removal.
[0037] Fig.13 Comparison of CE-SDS spectrum and RP-HPLC chromatogram of PEG-EPO DS sample. DETAILED DESCRIPTION
[0038] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.
[0039] Example 1 A method for detecting the purity of PEGylated recombinant human erythropoietin
[0040] 1 Experimental supplies
[0041] 1.1 Key experimental instruments are shown in Table 1.
[0042] Table 1 Experimental instruments
[0043] name factory model Capillary electrophoresis instrument Protein simple Maurice Constant temperature metal bath Eppendorf Mixer Ultra-trace UV detector Eppendorf BioSpectrometer
[0044] 1.2 Key reagents and consumables are shown in Table 2.
[0045] Table 2 Reagents and consumables
[0046]
[0047]
[0048] 1.3 Solution preparation
[0049] 1) 0.2M sodium dihydrogen phosphate
[0050] Weigh 1.5601 g NaH2PO4·2H2O, add 40 mL ultrapure water, stir to dissolve, make up to 50 mL with water, filter through a 0.22 μm filter membrane, and store at 4°C.
[0051] 2) 0.2M disodium hydrogen phosphate
[0052] Weigh 3.5814 g Na2HPO4·12H2O, add 40 mL ultrapure water, stir and dissolve, make up to 50 mL with water, filter through a 0.22 μm filter membrane, store at 25°C, and filter through a 0.22 μm filter membrane before use.
[0053] 3) 100 mM phosphate buffer (pH 6.5 ± 0.1, 31.4 mM disodium hydrogen phosphate, 68.6 mM sodium dihydrogen phosphate):
[0054] Use a pipette to transfer 1570 μL of 0.2 M disodium hydrogen phosphate, 3430 μL of 0.2 M sodium dihydrogen phosphate, and 5000 μL of ultrapure water, stir to dissolve, filter through a 0.22 μm filter membrane, and store at 4°C.
[0055] 4) 40 mM phosphate buffer (pH 7.5 ± 0.1, 6.4 mM disodium hydrogen phosphate, 33.6 mM sodium dihydrogen phosphate)
[0056] Use a pipette to transfer 3.2 mL of 0.2 M disodium hydrogen phosphate, 16.8 mL of 0.2 M sodium dihydrogen phosphate, and 80 mL of ultrapure water, stir to dissolve, filter through a 0.22 μm filter membrane, and store at 4°C for 3 months or at -20°C.
[0057] 5) Sample buffer 2X Sample buffer (pH 6.5, 12.56 mM disodium hydrogen phosphate, 27.44 mM sodium dihydrogen phosphate, 2% SDS)
[0058] Use a pipette to take 400 μL of 100 mM phosphate buffer (pH 6.5±0.1), 200 μL of 10% SDS solution, and 400 μL of ultrapure water, vortex to mix, and prepare before use.
[0059] 6) 25X internal standard
[0060] Remove the metal seal on the 25X internal standard lyophilized powder internal standard bottle, and then remove the rubber stopper; add 240μL 1XSample Buffer and mix by pipetting up and down.
[0061] 2 Experimental procedures
[0062] 2.1 Sample pretreatment
[0063] The sample was concentrated and replaced with enzyme digestion buffer with a target concentration of 2 mg / mL. The enzyme digestion buffer is suitable for the following enzyme digestion operations, as follows:
[0064] 2.1.1 Cleaning the ultrafiltration membrane: insert the Amicon ULtra-0.5mL (10KD) device into a centrifuge tube, add 450μL ultrapure water to the Amicon ULtra-0.5mL (10KD) device, and centrifuge at 14000rpm for 10min; discard the liquid in the lower centrifuge tube and aspirate the remaining water in the Amicon ULtra-0.5mL (10KD);
[0065] 2.1.2 Concentration: Take 450 μL of PEG-EPO test sample (from Ond Bio) into the Amicon ULtra-0.5mL (10KD) centrifuge tube treated in step 2.1.1, centrifuge at 14000rpm for 10-15min, (Note: if the sample concentration is ≤500μg / mL, the required sample volume exceeds 450μL, and this step may need to be repeated multiple times), discard the waste liquid at the bottom of the centrifuge tube, if there is only a small amount of waste liquid, this operation is not required;
[0066] 2.1.3 Solvent replacement: Take 400 μL of enzyme digestion buffer (40 mM PB (pH 7.5 ± 0.1)) and place it in the Amicon ULtra-0.5 mL (10KD) centrifuge tube treated in step 2.1.2, centrifuge at 14000 rpm for 10 min, discard the liquid in the lower centrifuge tube, and perform this step 3 times in total;
[0067] 2.1.4 Concentration determination: Insert the Amicon ULtra-0.5mL (10KD) device upside down into a new centrifuge tube, centrifuge at 1000rpm for 2min, take out the sample solution on the upper end of the ultrafiltration membrane in the ultrafiltration tube, use an ultra-micro UV detector to determine the concentration after concentration, and dilute with 40mM PB (pH7.5±0.1) to the target concentration of 2.0mg / mL.
[0068] 2.2 Sample N-glycan removal
[0069] The samples were digested with PNGase F enzyme.
[0070] 2.2.1 Optimization of PNGase F incubation time
[0071] Sample desugaring: Take 100 μL of the pretreated PEG-EPO (40 mM PB (pH 7.5 ± 0.1)) obtained in step 2.1 and put it into a 1.5 mL centrifuge tube. Add 4 μL PNGase F to the tube, mix well and centrifuge at 1,500 g for 2 min. Take the supernatant and incubate at 37°C for 2h / 4h / 6h / 16h (overnight) to obtain the N-desugared PEG-EPO.
[0072] Sample preparation: Take 80 μL of sample premix solution, add 20 μL of PEG-EPO test solution after N-sugar removal at 2.0 mg / mL, mix thoroughly, centrifuge at 2500 rpm for 5 minutes to remove bubbles, denature at 70°C for 10 minutes, cool naturally to room temperature, carefully draw about 80 μL from the upper layer and add it to the detection ELISA plate, centrifuge at 2000 rpm for 5 minutes using a flat centrifuge, and perform capillary gel electrophoresis analysis. The reference solution and blank sample (ultrapure water) are operated in the same way.
[0073] The sample premix solution formula is shown in Table 4.
[0074] The experimental results are shown in Table 3 and Figure 1 .
[0075] Figure 1 The electrophoretic patterns of PEG-EPO samples incubated with PNGase F for different time periods.
[0076] Table 3 Peak area ratio of each component in PEG-EPO samples after enzyme incubation for different time
[0077]
[0078]
[0079] Result analysis: As the incubation time of PNGase F increased, the area proportion of the main peak of the PEG-EPO sample gradually increased, while the area proportion of HMW gradually decreased; when the incubation time of PNGase F increased to 16h, the main peak purity of the sample was as high as 99.3%.
[0080] Experimental conclusion: Enzyme digestion incubation time ≥ 16h (overnight).
[0081] 2.3 Preparation of sample premix solution
[0082] The optimization of the sample premix solution system included: sample buffer volume, sample buffer pH, and removal of the 10kD internal standard.
[0083] 2.3.1 Optimization of Sample Buffer Volume
[0084] Use 1X Sample buffer and 2X Sample buffer respectively, add different volumes according to Table 4 and Table 5, prepare different sample premix solutions, increase the concentration of the sample in the premix solution, and maintain the final concentration of SDS at 2%.
[0085] (1) The formula of the premixed solution of 1X Sample buffer and the ratio of addition to the sample solution are shown in Table 4:
[0086] Table 4 1X Sample buffer premix solution formula and addition ratio to sample solution
[0087] Reagents Reagent addition amount 1X sample buffer 71μL 250mM NEM 5μL 25x Internal Standard 4μL Total volume 80μL
[0088] Take 80 μL of the sample premix solution and add 20 μL of 2.0 mg / mL N-glycan-depleted PEG-EPO solution to make the final concentration of the sample 0.4 mg / mL.
[0089] (2) The formula of the premixed solution of 2X Sample buffer and the ratio of addition to the sample solution are shown in Table 5:
[0090] Table 5 2X Sample buffer premix formula and addition ratio to sample solution
[0091] Reagents Reagent addition amount 2X Sample Buffer 44μL 250mM NEM 5μL 25x Internal Standard 1μL Total volume 50μL
[0092] Take 50 μL of the sample premix solution and add 50 μL of 2.0 mg / mL N-glycan-depleted PEG-EPO solution to make the final concentration of the sample 1.0 mg / mL.
[0093] The experimental results are shown in Figure 2 , Figure 3 and Table 6.
[0094] Figure 2 This is the electrophoresis spectrum of the PEG-EPO sample with 1X Sample buffer premix.
[0095] Figure 3 This is the electrophoresis spectrum of the PEG-EPO sample with 2X Sample buffer premix.
[0096] Table 6 Peak area percentage results of PEG-EPO samples in different sample buffers
[0097] Sample buffer Low molecular weight component (LMW)% Main peak% High molecular weight component (HMW)% 1X 0.1 99.3 0.5 2X 0.1 97.5 2.5
[0098] Result analysis: By comparing the two figures, it is found that by changing the volume of the Sample buffer and increasing the final concentration of the sample, the peak height of the sample can be significantly increased, and low-abundance fragments and aggregates can be detected.
[0099] Experimental conclusion: Prepare the sample premix according to the above method (2).
[0100] 2.3.2 Optimization of pH of Sample Buffer
[0101] 2XSample buffers with different pH values were selected, and the pH values were 6.5 and 9.0 respectively (obtained by adjusting the pH of 2XSample buffer at pH 6.5 with 0.2 M disodium hydrogen phosphate solution).
[0102] The experimental results are shown in Figure 4 and Table 7:
[0103] Figure 4 The electrophoresis patterns of PEG-EPO samples at different sample buffer pH values.
[0104] Table 7 Peak area percentage results of different sample buffer pH
[0105] Sample buffer pH Low molecular weight component (LMW)% Main peak% High molecular weight component (HMW)% 6.5 0.1 97.5 2.5 9.0 0.1 97.4 2.5
[0106] Result analysis: Sample buffers with pH values of 6.5 and 9.0 had no significant effect on the migration time, fragmentation and aggregation of PEG-EPO samples. In order to avoid the degradation effect of high pH on the samples, a sample buffer with a pH value of 6.5 was selected for subsequent experiments.
[0107] Experimental conclusion: Select Sample buffer with a pH value of 6.5.
[0108] 2.3.3 Removal of 10 kD internal standard
[0109] Sample preparation: In order to avoid the interference of the 10kD internal standard peak on the detection of possible protein impurities and fragment peaks, the sample premix solution was prepared according to Table 8.
[0110] Table 8 Sample premix solution (taking a single sample as an example)
[0111] Reagents Reagent addition amount 2X Sample Buffer 45μL 250mM NEM 5μL Total volume 50μL
[0112] The experimental results are shown in Figure 5 .
[0113] Figure 5 The electrophoresis spectrum of the PEG-EPO sample without the 10kD internal standard. Among them, Neg-Blank is the negative control group, PEG-EPO DS is the PEG-EPO stock solution (derived from Shandong Ande Biological, which is used for the preparation and filling of PEG-EPO DP after dilution and addition of excipients), and PEG-EPO DP is the PEG-EPO injection (a product of Shandong Ande Biological Preparations).
[0114] Result analysis: The system peak at 525 seconds has no effect on the sample fragment peak and can replace the 10kD internal standard peak for relative migration time and peak area calculation of internal standard correction.
[0115] The final premix solution formula is shown in Table 8. Note: In non-reduction experiments, NEM can be replaced with IAM; in reduction experiments, NEM needs to be replaced with BME.
[0116] 2.4 Sample preparation
[0117] The samples were denatured by incubation, and the denaturation conditions were: incubation at 70°C for 10 min, incubation at 95°C for 5 min, and incubation at 95°C for 10 min.
[0118] The details are as follows:
[0119] ① Mix 50 μL of the N-deglycosylated PEG-EPO prepared in step 2.2 and 50 μL of the sample premix solution prepared in step 2.3 in a 1.5 mL centrifuge tube to a total volume of 100 μL;
[0120] ② Place the 1.5 mL centrifuge tube from step ① in a centrifuge and centrifuge at 12000 rpm for 5 min (in order to prevent possible insoluble particles from affecting the protein denaturation process, if there are insoluble particles, the supernatant needs to be transferred to another 1.5 mL centrifuge tube for the next step);
[0121] ③ Place the 1.5 mL centrifuge tube containing the supernatant in a metal bath (or water bath) and incubate at different temperatures for different denaturation times;
[0122] ④Take out the 1.5mL centrifuge tube and cool it naturally to room temperature;
[0123] ⑤ Place the 1.5 mL centrifuge tube in a centrifuge, centrifuge at 12000 rpm for 5 min, and transfer 70 μL of the supernatant to a 96-well plate (avoid bubbles when adding liquid);
[0124] ⑥ Place the 96-well plate in a centrifuge and centrifuge at 2500 rpm for 5 minutes to remove bubbles and set aside;
[0125] ⑦Install the card box and perform sample testing.
[0126] The experimental results are shown in Figure 6 .
[0127] Figure 6 The electrophoresis patterns of PEG-EPO samples under different denaturation conditions.
[0128] Result analysis: It can be seen from the figure that there is no obvious difference in the electrophoresis spectrum of PEG-EPO samples under the three denaturing conditions of incubation at 70℃ for 10min, incubation at 95℃ for 5min, and incubation at 95℃ for 10min. The peak type of the electrophoresis spectrum remains consistent, indicating that 70℃ and 95℃ have no obvious effect on the non-reduced CE-SDS results of PEG-EPO samples. In order to reduce the possible impact of temperature on PEG-EPO samples, the lower 70℃ was selected for subsequent experiments.
[0129] Experimental conclusion: The denaturation condition is incubation at 70℃ for 10 minutes.
[0130] 2.5 Capillary gel electrophoresis (CE-SDS) analysis
[0131] Take the 96-well plate containing samples prepared in step 2.4 and place it in a capillary electrophoresis instrument to perform a capillary gel electrophoresis experiment. The capillary gel electrophoresis acquisition method is shown in Table 9.
[0132] Table 9 Collection method
[0133]
[0134]
[0135] 2.6 Data Analysis
[0136] The instrument's own software Compass for iCE or empower and other third-party software were used for integration, and the purity of the sample was calculated based on the peak area ratio of different components. All protein peaks (except PNGase F enzyme) after the internal standard peak in the sample spectrum were integrated and counted, and the peak area ratio of the main peak, i.e., the sample purity, was calculated according to the area normalization method.
[0137] 3 Experimental results
[0138] The experimental results are shown in Figure 7 .
[0139] Figure 7 CE-SDS electrophoresis patterns of PEG-EPO and EPO samples for application of the method of the present invention (from top to bottom: blank control, EPO, PEG-EPO).
[0140] Example 2 Performance measurement of the method of the present invention
[0141] (1) Exclusivity
[0142] The detection performance of the method of the present invention was investigated using a blank control solution (ultrapure water), a negative control solution (PNase F enzymatic hydrolysis solution without sample), and high temperature stability test samples placed for different times (prepared by placing PEG-EPO DS (Lot.: PE202101) at 45°C at 0W / 1W / 2W / 4W / 6W / 8W). The experimental results are shown in Figure 8 , Fig. 9 and Table 10.
[0143] Figure 8 Shown are the electrophoretic patterns of blank control solution, negative control solution and PEG-EPO DS sample solution.
[0144] Fig. 9 This is the electrophoresis spectrum of the high temperature degradation sample of PEG-EPO DS.
[0145] Table 10 Peak area percentage results of PEG-EPO DS high temperature degradation samples
[0146] Degradation conditions Low molecular weight component (LMW)% Main peak% High molecular weight component (HMW)% 45℃0W 0.1 98.8 1.1 45℃1W 0.4 97.9 1.7 45℃2W 0.6 96.6 2.9 45℃4W 0.9 93.4 5.7 45℃6W 1.2 90.4 8.4 45℃8W 2.1 86.5 11.4
[0147] Result analysis:
[0148] ① In the blank control solution, there is no visible interference peak;
[0149] ② In the negative control solution, except for PNGase F, there are no other interfering peaks;
[0150] ③At 45℃, the main peak area percentages of 0W, 1W, 2W, 4W, 6W and 8W all decreased to varying degrees, while the peak area percentages of fragment peaks and aggregates increased with the extension of degradation time, indicating that this method can identify the changing trend of non-reduced CE-SDS purity of forced degradation samples.
[0151] (2) Repeatability
[0152] Under the same operating conditions and in the same sequence, six samples with a concentration of 2.0 mg / mL were repeatedly prepared, each sample was injected once, and the RSD% of the area percentage of the main peak of the six injections was calculated. The experimental results are shown in Table 11.
[0153] Table 11 Peak area and peak area ratio results of 6 repeated samples
[0154]
[0155] Result analysis: The RSD% of the sample's main peak area, total peak area, and main peak area ratio were 3.0%, 3.1%, and 0.2%, respectively, all less than 5%.
[0156] Experimental conclusion: This method has good repeatability.
[0157] (3) Linear investigation
[0158] The detection linearity of the method of the present invention was investigated when the initial sample concentration was 1.0-2.5 mg / mL. Fig.10 , Fig.11 and Table 12.
[0159] Fig.10 It is the linear result between the initial concentration of the sample and the peak area of the main peak of the sample.
[0160] Fig.11 It is the linear result of the initial concentration of the sample and the total peak area of the sample.
[0161] Table 12 Peak area and peak area ratio of samples with different concentrations
[0162] Conc.(mg / mL) Main peak area Total peak area LMW% Main peak% HMW% 1.0 817.9 831.2 NA 98.4 1.6 1.8 1300.5 1320.8 NA 98.5 1.5 2.0 1456.0 1480.7 NA 98.3 1.7 2.2 1592.2 1616.9 NA 98.5 1.5 2.5 1809.1 1840.7 NA 98.3 1.7
[0163] Result analysis: In the range of 1.0-2.5 mg / mL sample initial concentration, the sample concentration is linearly related to the main peak area and the total peak area. 2 They are 0.9974 and 0.9972 respectively, both greater than 0.99.
[0164] Experimental conclusion: The linearity is good within the range of 1.0-2.5 mg / mL of initial sample concentration.
[0165] In summary, the specificity investigation test results prove that the detection method established by the present invention has good specificity; the repeatability investigation test results prove that the detection method established by the present invention has good repeatability; the linear range investigation test results prove that the detection method established by the present invention has good linearity within the range of 1.0-2.5 mg / mL of the initial sample concentration.
[0166] Comparative Example 1
[0167] Compared with Example 1, this comparative example does not include step 2.2 "sample N-glycan removal treatment", and the remaining experimental steps are the same as those in Example 1, and the samples used are the same. The experimental results are shown in Fig.12 .
[0168] Fig.12 Conventional CE-SDS electrophoresis patterns of PEG-EPO and EPO samples without N-glycoside removal (from top to bottom: blank control, EPO, PEG-EPO).
[0169] As can be seen from the figure, PEG-EPO and EPO both have only one peak, and the peak shapes are similar without significant difference, although the molecular weights of the two differ by one fold (60 kD and 30 kD, respectively); and both have a wide peak width and a low peak height, which are quite different from the normal protein peak shape, indicating that the normal CE-SDS spectrum cannot be detected when the sample is not de-N-sugared.
[0170] Comparative Example 2
[0171] The same PEG-EPO DS sample was tested using reverse phase liquid chromatography (RP-HPLC) and the method of the present invention. The experimental results are shown in Fig.13 and Table 13.
[0172] Fig.13 Comparison of CE-SDS spectrum and RP-HPLC chromatogram of PEG-EPO DS sample.
[0173] from Fig.13 It can be seen that both CE-SDS and RP-HPLC methods can separate high molecular weight impurity peaks, and comparing the purity data measured by the two methods, the purity measured by CE-SDS is 98.4%, and the purity measured by RP-HPLC is 99.0%. The two are consistent, indicating that the method of the present invention can achieve the detection accuracy of the known methods in the art.
[0174] Table 13 Comparison of purity of PEG-EPO by different detection methods
[0175] Detection Methods Main peak area% Impurity peak area% CE-SDS 98.4 1.6 RP-HPLC 99.0 1.0
[0176] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for detecting the purity of PEGylated recombinant human erythropoietin, characterized in that: The following steps are involved: (1) Using PNGase F enzyme to remove N-glycans from the PEGylated recombinant human erythropoietin to obtain N-glycan-deglycosylated PEG-EPO; (2) adding SDS to denature the N-glycan-depleted PEG-EPO; (3) Perform capillary gel electrophoresis and calculate the purity of the sample based on the peak area ratio of different components.
2. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 1, wherein: Before step (1), the method further includes pre-treating the PEGylated recombinant human erythropoietin to be tested to obtain pre-treated PEG-EPO; the pre-treatment is to concentrate the PEGylated recombinant human erythropoietin to be tested and replace the solution with an enzyme cleavage buffer.
3. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 2, wherein: The concentration is carried out using an ultrafiltration membrane; and / or The enzyme digestion buffer was 40 mM PB at pH 7.5±0.
1.
4. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 1, wherein: In step (1), the N-glycan removal treatment is to mix PNGase F enzyme with the PEGylated recombinant human erythropoietin to be tested and centrifuge, and take the supernatant and incubate at 37±1°C for ≥16h.
5. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 1, wherein: In step (2), the N-glycan-depleted PEG-EPO is mixed with the sample premix solution and then incubated for denaturation; The sample premix solution consists of the following components in the following volumes: 9 parts 2X Sample Buffer; 1 part 250mM NEM; The formula of the 2X Sample Buffer is: 12.56 mM disodium hydrogen phosphate, 27.44 mM sodium dihydrogen phosphate, 2% SDS, pH 6.5-9.
0.
6. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 5, wherein: The pH value is 6.
5.
7. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 5, wherein: After the N-glycan-depleted PEG-EPO was mixed with the sample premix solution, the final concentration of the N-glycan-depleted PEG-EPO was 1.0 mg / mL.
8. The method for detecting the purity of PEGylated recombinant human erythropoietin according to any one of claims 1 to 7, characterized in that: In step (2), the incubation and denaturation conditions are: 70-90° C., incubation for 5-10 minutes.
9. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 8, wherein: The incubation and denaturation conditions are: 70° C., incubation for 10 min.
10. The method for detecting the purity of PEGylated recombinant human erythropoietin according to claim 1, wherein: In step (3), the collection method of the capillary gel electrophoresis is as follows: injection: 4600V, 20s; Separation: 5750V, 30min.