CN118243763A - Optimized CE-SDS purity detection method - Google Patents
Optimized CE-SDS purity detection method Download PDFInfo
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- CN118243763A CN118243763A CN202211657026.4A CN202211657026A CN118243763A CN 118243763 A CN118243763 A CN 118243763A CN 202211657026 A CN202211657026 A CN 202211657026A CN 118243763 A CN118243763 A CN 118243763A
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- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 8
- 238000005251 capillar electrophoresis Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 239000012723 sample buffer Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 230000029936 alkylation Effects 0.000 claims description 2
- 238000005804 alkylation reaction Methods 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 206010040844 Skin exfoliation Diseases 0.000 claims 1
- 230000035618 desquamation Effects 0.000 claims 1
- 229940125645 monoclonal antibody drug Drugs 0.000 abstract description 14
- 102000014128 RANK Ligand Human genes 0.000 abstract description 4
- 108010025832 RANK Ligand Proteins 0.000 abstract description 4
- 238000003908 quality control method Methods 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 2
- 229940126585 therapeutic drug Drugs 0.000 abstract 1
- 238000011156 evaluation Methods 0.000 description 16
- 238000012360 testing method Methods 0.000 description 16
- 239000003814 drug Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000012937 correction Methods 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- Peptides Or Proteins (AREA)
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Abstract
The invention discloses an optimized CE-SDS purity detection method and application thereof. The detection method of the invention is used for determining the purity of the anti-RANKL monoclonal antibody drug, and verifying the specificity, linearity, range, accuracy and precision, which has important significance for research and development and quality control of the RANKL targeted therapeutic drug.
Description
Technical Field
The method relates to the technical field of biology, in particular to an optimized non-reducing CE-SDS purity detection method and application thereof in monoclonal antibody purity detection.
Background
Common methods for detecting monoclonal antibody purity include SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and SEC-HPLC (molecular sieve exclusion chromatography high performance liquid chromatography). SDS-PAGE is long in time consumption, complex in operation and poor in repeatability, and the purity of the medicine is difficult to calculate accurately; the SEC-HPLC has low separation degree, limited detection range, larger size range of aggregates formed by protein drugs, possibility of filtering out most of the polymers by SEC chromatographic columns and inaccurate detection result. The CE-SDS is a method for detecting the purity of the antibody under the denaturation condition, the principle of the method is similar to that of SDS-PAGE, but compared with SDS-PAGE, the CE-SDS utilizes capillary electrophoresis to separate samples, and fluorescent dye is added into the samples for analysis and detection, so that the method has the advantages of small sample loading amount, large flux, high resolution, accurate quantification and the like.
The antibody medicine has large molecular mass, complex structure and high heterogeneity, and the detection method on the market at present is not completely suitable for detecting all monoclonal antibodies, so new requirements and challenges are provided for characterization analysis and quality control of monoclonal antibody medicines.
Disclosure of Invention
The invention aims to provide an optimized non-reducing CE-SDS purity detection method, in particular to a detection method for the purity of an anti-RANKL monoclonal antibody drug, which has the advantages of simple operation, rapid analysis, accurate result, good precision, stable and reliable detection result and batch detection.
In one aspect, the invention provides an optimized CE-SDS purity detection method comprising the steps of:
(1) Sample solution preparation: preparing a protein sample to be tested, namely a buffer solution and an alkylation solution;
(2) Loading: incubating the sample solution prepared in the step (1) at 65-75 ℃ for 2-10min, mixing uniformly by vortex, centrifuging, taking the supernatant, and centrifuging;
(3) Analysis: analyzing the centrifuged supernatant obtained in step (2) by using a capillary electrophoresis apparatus so as to obtain the main peak purity.
Further, according to an embodiment of the present invention, the preparation steps of the sample solution of the step (1) are as follows: taking 100ug of protein sample to be detected, adding 5 ul IAM solution, and supplementing the sample buffer solution to 100 ul; the IAM concentration was 800mM.
Further, according to an embodiment of the present invention, the final concentration of the sample solution of the step (1) is 0.5mg/ml to 1.5mg/ml.
Further, according to an embodiment of the present invention, the final concentration of the sample solution of the above step (1) is 1.0mg/ml.
Further, according to an embodiment of the present invention, the protein sample of step (1) above is an anti-RANKL monoclonal antibody; preferably, the protein sample is Desumab
Further, according to an embodiment of the present invention, in the step (2) above: incubating the sample solution prepared in the step (1) for 5min at 70 ℃, and centrifuging after vortex mixing uniformly, taking the supernatant and centrifuging. .
Further, according to an embodiment of the present invention, the capillary electrophoresis apparatus in the step (3) is a Maurice fully-automatic protein capillary electrophoresis apparatus.
Further, according to an embodiment of the present invention, the analysis conditions of the above step (3): sample feeding conditions are 15s and 4600V, and separation conditions are 40min and 5750V.
Further, according to an embodiment of the present invention, the above method is a detection method under non-reducing conditions.
In a second aspect, the invention provides the use of the above detection method in the detection of monoclonal antibody purity.
The invention has the following beneficial technical effects:
The invention establishes a non-reducing CE-SDS (NR CE-SDS) CE-SDS detection method aiming at the RANKL monoclonal antibody medicine, which can meet the requirements on specificity, linearity, range, accuracy, precision and the like in the verification process of the method, is a non-reducing CE-SDS detection method aiming at the RANKL monoclonal antibody medicine with high accuracy, high precision and good stability, can simply, quickly and efficiently detect the medicine purity, and has important significance on the aspects of research and development, quality control and the like of the RANKL targeted therapeutic medicine.
Drawings
FIG. 1 shows the assay specificity evaluation of the monoclonal antibody drug NR CE-SDS against an embodiment of the present invention;
FIG. 2 shows the linearity and range evaluation of monoclonal antibody drug NR CE-SDS detection against an embodiment of the present invention;
FIG. 3 shows the assay reproducibility evaluation of the monoclonal antibody drug NR CE-SDS against an embodiment of the present invention;
FIG. 4 shows an intermediate precision evaluation of the monoclonal antibody drug NR CE-SDS assay for an embodiment of the present invention;
FIG. 5 shows evaluation of durability of detection of a monoclonal antibody drug NR CE-SDS against an embodiment of the present invention.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art
EXAMPLE 1 monoclonal antibody purity detection
The detection method comprises the following steps of the main materials for the test shown in table 1, the main instruments for the test shown in table 2, and the samples for the test shown in table 3 (the amino acid sequence of the Deshu Shan Kangchong chain of the anti-RANKL monoclonal antibody drug is shown as SEQ ID NO.1, the amino acid sequence of the light chain is shown as SEQ ID NO.2, and the specific preparation method is referred to as CN 103232539B).
(1) 100Ug of protein Sample was taken and 5 ul of 800mM IAM solution was added and the Sample buffer (Sample buffer) was made up to 100 ul so that the final concentration of protein Sample was 1.0mg/ml.
(2) The sample solution is heated and incubated for 5min at 70 ℃, cooled to room temperature, and then is uniformly mixed by vortex and centrifuged for 5min at 12000 rpm. 75 μl of sample supernatant was placed in 96-well plates, centrifuged at 1000g for 5min to ensure no air bubbles in the sample wells, and then prepared for on-machine separation.
(3) Sample introduction conditions: 15s,4600v; separation conditions: 40min,5750V.
(4) And (3) calculating results: according to the area normalization method, the ratio of the corrected peak area of a certain peak is calculated by dividing the corrected peak area of the certain peak by the sum of the corrected peak areas of all peaks in an integral window, and the corrected peak area percentage of the main peak of IgG is used as the purity result of the monoclonal antibody drug.
Table 1 main materials for testing
Table 2 main instrument for test
| Instrument name | Manufacturing factories | Model number |
| Full-automatic protein capillary electrophoresis apparatus | Protein Simple | Maurice |
| Constant temperature mixing instrument | Eppendorf | Thermo Mixer C |
| Desk type centrifugal machine | Beckmann Coulter Co.Ltd | J-15R |
| Centrifugal machine | Beckmann Coulter Co.Ltd | Microfuge 20R |
Table 3 samples for testing
| Name of the name | Concentration of | Lot number | Manufacturing factories |
| Deshu monoclonal antibody working reference | 10.1mg/ml | RS201905002 | Jiangsu Taikang biological medicine Co., ltd |
SEQ ID NO.1: dehu Shan Kangchong chain amino acid sequence
SEQ ID NO.2: deshu monoclonal antibody light chain amino acid sequence
Example 2 test method evaluation test
2.1 Specificity evaluation test
The reference Desmomab and ddH2O (blank control) were used as materials, and the detection was performed by the detection method of example 1, and the detection result is shown in FIG. 1, and no interference peak appears in the blank sample. The experimental result shows that the invention has good specificity.
2.2 Linear and Range evaluation test
Taking reference Desmomab as a material, respectively taking a proper amount of reference, adding 5 mu l of 800mM IAM solution, and supplementing the sample buffer solution to 100 mu l, so that the final concentration of the protein sample is 0.5mg/ml, 0.75mg/ml, 1.0mg/ml, 1.25mg/ml and 1.5mg/ml, and respectively preparing 3 samples for each concentration. The linear regression was performed using the detection method of example 1, with reference concentration X (mg/ml) as the abscissa and main peak area Y as the ordinate, to determine the linear range. As shown in FIG. 2 and Table 4, the main peak area increases linearly with the increase of the sample concentration, and the linear correlation coefficient R 2 is 1.00, which indicates that the final concentration of the sample is in the range of 0.5mg/ml to 1.5mg/ml (50% -150%), and the linearity of the method is good.
TABLE 4 anti-RANKL monoclonal antibody drug NR CE-SDS test linearity and range evaluation results summary table
| Final concentration of sample (mg/ml) | Peak area 1 | Peak area 2 | Peak area 3 |
| 0.50 | 1617.1 | 1792.5 | 1782.5 |
| 0.75 | 2530.5 | 2657.8 | 2642.4 |
| 1.00 | 3391.8 | 3519.7 | 3536.7 |
| 1.25 | 4296.0 | 4385.5 | 4335.9 |
| 1.50 | 5172.3 | 5271.8 | 5207.3 |
2.3 Accuracy evaluation test
The detection method using reference Desmomab as material was the same as in example 1. The detection results are shown in Table 5, the recovery rate of each concentration sample is in the range of 90% -110%, and the method has good accuracy.
TABLE 5 evaluation results of anti-RANKL monoclonal antibody drug NR CE-SDS test accuracy summary
2.4 Precision evaluation test
The precision of the invention is evaluated by taking reference Deshu monoclonal antibody as a material. Firstly, adopting the detection method steps, preparing 6 samples in parallel for analysis, and examining the repeatability of the method; the above procedure was then repeated 3 times (6 samples were prepared in parallel) on different days to investigate the intermediate precision of the procedure.
The detection results of the 6 parallel samples are shown in fig. 3 and table 6, the RSD of the area percentage of the main peak correction peak is lower than 2.0%, and the RSD of the area percentage of the degradation peak correction peak is lower than 20.0%, which shows that the method of the invention has high repeatability.
The detection results of 3 parallel experiments carried out in 3 days are shown in fig. 4 and table 7, wherein the RSD of the area percentage of the main peak correction peak is lower than 2.0%, and the RSD of the area percentage of the degradation peak correction peak is lower than 20.0%, which indicates that the intermediate precision of the method is high.
TABLE 6 anti-RANKL monoclonal antibody drug NR CE-SDS test reproducibility evaluation results summary table
TABLE 7 intermediate precision evaluation results summary of anti-RANKL monoclonal antibody drug NR CE-SDS detection
2.5 Durability evaluation test
And (3) taking the reference Deshu monoclonal antibody as a material, and detecting at time points of 0h and 24h of sample placement by using the detection method. As shown in FIG. 5 and Table 8, the detection results show that the RSD of the area percentage of the main peak correction peak is lower than 2.0%, and the RSD of the area percentage of the degradation peak correction peak is lower than 20.0%, which indicates that the sample can be placed for 24 hours.
TABLE 8 evaluation results of anti-RANKL monoclonal antibody drug NR CE-SDS test durability summary
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. An optimized CE-SDS purity detection method comprises the following steps:
(1) Sample solution preparation: preparing a protein sample to be tested, namely a buffer solution and an alkylation solution;
(2) Loading: incubating the sample solution prepared in the step (1) at 65-75 ℃ for 2-10min, mixing uniformly by vortex, centrifuging, taking the supernatant, and centrifuging;
(3) Analysis: analyzing the centrifuged supernatant obtained in step (2) by using a capillary electrophoresis apparatus so as to obtain the main peak purity.
2. The method of claim 1, wherein the sample solution of step (1) is prepared by the steps of: taking 100ug of protein sample to be detected, adding 5 ul IAM solution, and supplementing the sample buffer solution to 100 ul; the IAM concentration was 800mM.
3. The method according to claim 1, wherein the final concentration of the sample solution in the step (1) is 0.5mg/ml to 1.5mg/ml.
4. The method of claim 1, wherein the final concentration of the sample solution in step (1) is 1.0mg/ml.
5. The assay of claim 1, wherein the protein sample of step (1) is an anti-RANKL monoclonal antibody; preferably, the protein sample is desquamation.
6. The method of claim 1, wherein in step (2): incubating the sample solution prepared in the step (1) for 5min at 70 ℃, and centrifuging after vortex mixing uniformly, taking the supernatant and centrifuging.
7. The method of claim 1, wherein the capillary electrophoresis apparatus of step (3) is a Maurice fully automated protein capillary electrophoresis apparatus.
8. The method of claim 1, wherein the analysis conditions of step (3) are: sample feeding conditions are 15s and 4600V, and separation conditions are 40min and 5750V.
9. The method of claim 1, wherein the method is a detection method under non-reducing conditions.
10. Use of the detection method according to any one of claims 1-9 for the detection of monoclonal antibody purity.
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