CN117347519A - Liquid chromatography method for detecting content of residual SN38 small molecules in antibody coupled drug - Google Patents
Liquid chromatography method for detecting content of residual SN38 small molecules in antibody coupled drug Download PDFInfo
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- 229940079593 drug Drugs 0.000 title claims abstract description 41
- 239000003814 drug Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 34
- -1 SN38 small molecules Chemical class 0.000 title claims abstract description 22
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 17
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 11
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000010790 dilution Methods 0.000 claims abstract description 7
- 239000012895 dilution Substances 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 6
- 229940123780 DNA topoisomerase I inhibitor Drugs 0.000 claims abstract description 4
- 239000000365 Topoisomerase I Inhibitor Substances 0.000 claims abstract description 4
- 230000008878 coupling Effects 0.000 claims abstract description 3
- 238000010168 coupling process Methods 0.000 claims abstract description 3
- 238000005859 coupling reaction Methods 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000011084 recovery Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 11
- 239000003085 diluting agent Substances 0.000 claims description 10
- 239000012086 standard solution Substances 0.000 claims description 10
- 238000002372 labelling Methods 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 6
- 239000010413 mother solution Substances 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
- 239000004475 Arginine Substances 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 229950008882 polysorbate Drugs 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 238000000825 ultraviolet detection Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 1
- 239000012898 sample dilution Substances 0.000 claims 1
- 150000003384 small molecules Chemical class 0.000 abstract description 11
- 238000001556 precipitation Methods 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000005119 centrifugation Methods 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 46
- 239000000611 antibody drug conjugate Substances 0.000 description 33
- 229940049595 antibody-drug conjugate Drugs 0.000 description 33
- 239000012071 phase Substances 0.000 description 25
- 230000014759 maintenance of location Effects 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 9
- 239000012452 mother liquor Substances 0.000 description 8
- 235000010290 biphenyl Nutrition 0.000 description 5
- 239000004305 biphenyl Substances 0.000 description 5
- 125000006267 biphenyl group Chemical group 0.000 description 5
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 4
- 239000012496 blank sample Substances 0.000 description 3
- 229940127089 cytotoxic agent Drugs 0.000 description 3
- 239000002254 cytotoxic agent Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012434 mixed-mode chromatography Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/89—Inverse chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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Abstract
The invention discloses a liquid chromatography for detecting the content of residual SN38 small molecules in an antibody coupled drug, which is characterized in that an ADC sample of the antibody coupled drug is directly injected or injected after dilution, and the content of SN38 of a free topoisomerase I inhibitor in the sample is detected by adopting a reversed phase chromatography; the ADC sample is an antibody coupling drug coupled with SN 38; the mobile phase A of the reversed phase chromatography is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution. The advantages are that: (1) Aiming at the ADC sample containing residual SN38 small molecules, the sample can be directly injected with or without dilution, and complex pretreatment such as precipitation, centrifugation and the like is not needed, so that the detection time is greatly shortened; (2) The method avoids incomplete precipitation and small molecule hydrolysis caused by pretreatment, thereby having high sensitivity; (3) The method has good specificity, repeatability and linearity.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and relates to a method for detecting residual small molecules in an antibody-coupled drug by adopting a liquid chromatography, in particular to a liquid chromatography for detecting the content of residual SN38 small molecules in the antibody-coupled drug.
Background
The antibody-drug conjugate (ADC) combines the high specificity of monoclonal antibody drugs with the high activity of small molecule cytotoxic drugs, so as to improve the targeting of tumor drugs and reduce the toxic and side effects. The small molecule cytotoxic drug is coupled with the protein structure through a linker (linker) structure to prepare the ADC. CL2A is a common linker, topoisomerase I inhibitor SN38 is a common small molecule cytotoxic drug, and CL2A and SN38 are first linked to give the drug linker CL2A-SN38 (drug-linker). In the ADC preparation process, the antibody firstly opens disulfide bonds through a reducing agent and further reacts with excessive CL2A-SN 38; by purification means, excess CL2A-SN38 is removed, but a small amount of free CL2A-SN38 may remain, causing potential toxic side reactions. Therefore, the content detection of residual CL2A-SN38 is a key quality parameter of ADC drugs.
In the prior art, high performance liquid chromatography is often adopted to detect the content of residual small molecules of ADC drugs. And adding excessive precipitant into the ADC sample, precipitating, centrifuging to obtain supernatant containing the small-molecule cytotoxic drug dissolved therein, and quantitatively analyzing by a reverse chromatographic column. The disadvantages are: 1. the pretreatment steps such as precipitation centrifugation are long; 2. the precipitant is generally composed of organic solvent and salt solution according to different proportions, and the screening step of the precipitant is long in time consumption and easy to cause incomplete precipitation. For CL2A-SN38, we found in the preliminary experiments that significant small molecule hydrolysis occurred during the pre-precipitation treatment step, resulting in a complex liquid-phase profile.
Another laboratory detection method suitable for CL2A-SN38 is Mixed mode chromatography, and Supelco SUPELCOSIL Hisep HPLC chromatographic column is used for realizing hydrophilic macromolecules andefficient separation of hydrophobic small molecules. The method does not require a pre-precipitation treatment and is therefore more efficient. But currently the Hisep column supplier "MilliporeSigma TM "no longer available, there is a strong need to develop an assay more suitable for CL2A-SN 38.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the defects of the prior art, a method which does not need pre-precipitation treatment and can directly sample and detect an ADC sample containing free SN38 is obtained, and meanwhile, good sensitivity, specificity and linearity are ensured; in view of this, the present invention provides liquid chromatography for detecting the residual SN38 small molecule content in antibody-conjugated drugs.
The technical scheme is as follows: the liquid chromatography method for detecting the content of residual SN38 small molecules in the antibody coupled drug comprises the steps of directly injecting or diluting an ADC sample of the antibody coupled drug, and then injecting, and detecting the content of SN38 of a free topoisomerase I inhibitor in the sample by adopting a reversed phase chromatography method; the ADC sample is an antibody coupling drug coupled with SN 38; the mobile phase A of the reversed phase chromatography is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution; the elution gradient of the reverse phase chromatography is as follows:
| time/minute | Mobile phase a ratio/% | Mobile phase B ratio/% |
| 0 | 90 | 10 |
| 19 | 62.5 | 37.5 |
| 19.1 | 0 | 100 |
| 23 | 0 | 100 |
| 23.1 | 90 | 10 |
| 25 | 90 | 10 |
。
Preferably, the SN38 small molecule is a SN 38-based drug linker, including CL2A-SN38, CL2E-SN38, MC-VC-PAB-SN38. Accordingly, the antibody conjugated drug ADC is not limited to various antibodies, nor to the kind of linker.
Preferably, the diluent for diluting the ADC sample adopts at least one of water, methanol, acetonitrile, ethanol and isopropanol.
Preferably, the diluent is a mixed solution of acetonitrile and water in a volume ratio of 25:75.
Preferably, the buffer of the ADC sample is not particularly limited, and may be any of various buffers well known to those skilled in the art, preferably at least one of histidine, arginine, sucrose, trehalose, and polysorbate.
Preferably, the column of the reverse phase chromatography is a large pore size reverse phase liquid chromatography column suitable for protein separation, such as: agilent Advance Bio RP-mAb chromatographic columns of phenyl-bonded phase; the wavelength of ultraviolet detection is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 mu L, and the column temperature is 25 ℃.
Preferably, the method comprises the step of drawing a linear standard curve of the SN38 small molecule standard, comprising the following steps:
(1) Dissolving an SN38 small molecular standard substance by using dimethylacetamide DMA to prepare a mother solution with the concentration of 10 mM;
(2) Preparing a mother solution prepared in the step (1) into a linear standard solution by adopting a diluent, wherein the concentration is as follows:
the method comprises the steps of carrying out a first treatment on the surface of the Wherein the diluent is at least one of water, methanol, acetonitrile, ethanol and isopropanol.
Preferably, when the mixed solution of acetonitrile and water in the volume ratio of 50:50 is used for dilution, a linear equation of a standard curve drawn by taking the concentration of the standard solution as an abscissa and the chromatographic peak area as an ordinate is as follows: y= 72.677x-4.5826, r 2 = 0.9991; when the mixed solution of acetonitrile and water with the volume ratio of 25:75 is used for dilution, the linear equation of the drawn standard curve is as follows: y= 88.2245x-4.5635, r 2 ＝0.9991。
Preferably, the specific steps of the method include:
s1, drawing a standard curve
Accurately weighing an SN38 small molecular standard substance, and dissolving by adopting dimethylacetamide DMA to prepare a mother solution; then gradually diluting with a diluent to prepare a linear standard solution, sampling and eluting, and preparing a linear standard curve by taking the concentration of the standard solution as an abscissa and the chromatographic peak area as an ordinate to obtain a linear equation;
s2, detecting SN38 content in antibody coupled drug ADC sample
And directly sampling or diluting an antibody-coupled drug ADC sample, sampling, eluting, reading chromatographic peak area, and calculating the concentration of SN38 in the sample and the labeling recovery rate according to the linear equation obtained in S1, wherein the labeling recovery rate/% = actual measurement plus scalar/theoretical plus scalar multiplied by 100%.
Preferably, the method has a standard recovery of 80-120%.
The beneficial effects are that: (1) Aiming at the ADC sample containing residual SN38 small molecules, the sample can be directly injected with or without dilution, and complex pretreatment such as precipitation, centrifugation and the like is not needed, so that the detection time is greatly shortened; (2) The method avoids incomplete precipitation and small molecule hydrolysis caused by pretreatment, thereby having high sensitivity; (3) The method has good specificity, repeatability and linearity.
Drawings
FIG. 1 is a sample detection chromatogram as described in example 1;
FIG. 2 is a graph showing the standard curve prepared in example 2;
FIG. 3 is a graph showing the standard curve prepared in example 3;
FIG. 4 is a graph showing the results of the sample test of example 4;
FIG. 5 is a sample detection chromatogram as described in example 5.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
(1) Preparation of molecular standard stock solution
The CL2A-SN38 standard 4.08mg was precisely weighed and dissolved in DMA to give 10mM Cl2A-SN38 mother liquor.
(2) Sample preparation
And taking a proper amount of ADC sample, and adding a small amount of CL2A-SN38 mother liquor to obtain a mixed solution of ADC and CL2A-SN38 for sample injection.
(3) Chromatographic conditions:
mobile phase a was 0.1% aqueous trifluoroacetic acid and mobile phase B was 0.1% acetonitrile trifluoroacetic acid, elution gradients are as set forth in the following table:
| time (minutes) | Mobile phase A ratio (%) | Mobile phase B ratio (%) |
| 0 | 90 | 10 |
| 28 | 50 | 50 |
| 28.5 | 0 | 100 |
| 29.5 | 0 | 100 |
| 30 | 90 | 10 |
| 35 | 90 | 10 |
The chromatographic column used was advanced Bio RP-mAb Diphenyl (2.1X100 mm,3.5 μm). The wavelength of the ultraviolet detector is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 mu L, and the column temperature is 25 ℃.
(4) Analysis of results:
the spectrogram is shown in figure 1. The retention time of ADC protein peak (figure 1: ADC peak) is 20-31min, the retention time of Cl2A-SN38 small molecular peak (figure 1: drug peak) is 15-17min, the retention time of residual CL2A-SN38 related peak (figure 1:drug relatedpeak) in the ADC sample is 5.5-6min, and the separation degree of the small molecular main peak and the protein is good. Under the existing gradient condition, the ADC is slow to elute, and the peak span is wider.
Example 2
(1) Preparation of molecular standard stock solution
The CL2A-SN38 standard 4.08mg was precisely weighed and dissolved in DMA to give 10mM Cl2A-SN38 mother liquor.
(2) Standard curve solution preparation
CL2A-SN38 mother liquor was formulated as a series of linear standard solutions using acetonitrile: the aqueous solution of water (50:50) was serially diluted. The concentrations are set forth in the following table:
| standard yeast solution | Concentration (mu M) |
| SD1 | 10 |
| SD2 | 5 |
| SD3 | 2 |
| SD4 | 1 |
| SD5 | 0.5 |
| SD6 | 0.2 |
| SD7 | 0.1 |
(3) Chromatographic conditions:
mobile phase a was 0.1% aqueous trifluoroacetic acid and mobile phase B was 0.1% acetonitrile trifluoroacetic acid, elution gradients are as set forth in the following table:
| time (minutes) | Mobile phase A ratio (%) | Mobile phase B ratio (%) |
| 0 | 90 | 10 |
| 19 | 62.5 | 37.5 |
| 19.1 | 0 | 100 |
| 23 | 0 | 100 |
| 23.1 | 90 | 10 |
| 25 | 90 | 10 |
The chromatographic column used was advanced Bio RP-mAb Diphenyl (2.1X100 mm,3.5 μm). The wavelength of the ultraviolet detector is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 mu L, and the column temperature is 25 ℃.
(4) And (3) calculating results: the concentration of the CL2A-SN38 standard curve solution is taken as an abscissa, and the chromatographic peak area is taken as a vertical axis to obtain a linear standard curve.
The linear equation is y= 72.677x-4.5826, r 2 = 0.9991; the spectrogram is shown in figure 2. FIG. 2 is a standard curve overlay, from bottom to top, SD1-SD7, blank solvents, respectively. The blank solvent has no interference peak at the peak position of CL2A-SN38, and the method has good specificity. The retention time of the small molecular peak is 10-16.5min, and the main peak has obvious front peak.
Example 3
(1) Preparation of small molecular standard stock solution
The CL2A-SN38 standard 4.08mg was precisely weighed and dissolved in DMA to give 10mM Cl2A-SN38 mother liquor.
(2) Standard curve solution preparation
CL2A-SN38 mother liquor was formulated as a series of linear standard solutions using acetonitrile: the aqueous solution of water (25:75) was serially diluted. The concentrations are set forth in the following table:
| standard yeast solution | Concentration (mu M) |
| SD1 | 10 |
| SD2 | 5 |
| SD3 | 2 |
| SD4 | 1 |
| SD5 | 0.5 |
| SD6 | 0.2 |
| SD7 | 0.1 |
(3) Chromatographic conditions:
mobile phase a was 0.1% aqueous trifluoroacetic acid and mobile phase B was 0.1% acetonitrile trifluoroacetic acid, elution gradients are as set forth in the following table:
| time (minutes) | Mobile phase A ratio (%) | Mobile phase B ratio (%) |
| 0 | 90 | 10 |
| 19 | 62.5 | 37.5 |
| 19.1 | 0 | 100 |
| 23 | 0 | 100 |
| 23.1 | 90 | 10 |
| 25 | 90 | 10 |
The chromatographic column used was advanced Bio RP-mAb Diphenyl (2.1X100 mm,3.5 μm). The wavelength of the ultraviolet detector is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 mu L, and the column temperature is 25 ℃.
(4) And (3) calculating results:
the blank solution did not interfere at the peak of CL2A-SN 38. The concentration of the CL2A-SN38 standard curve solution is taken as an abscissa, and the chromatographic peak area is taken as a vertical axis to obtain a linear standard curve. The linear equation is y= 88.2245x-4.5635, r 2 0.9991, the spectrum is shown in fig. 3, fig. 3 is a standard curve chart, SD1-SD7 from bottom to top, and the blank solvent. The retention time of the small molecular peak is 15-17min, the peak shape is symmetrical, and compared with the example 2, the peak shape is obviously improved. The blank solvent has no interference peak at the peak position of CL2A-SN38, and the method has good specificity.
The calculation results are shown in the following table:
linear equation R 2 >0.99, the recovery rate of each concentration point of the standard curve solution is 80-120%, which indicates that the method has good linearity.
(5) Repeatability verification
6 SD4 solutions were arranged in parallel and the calculation results are shown in Table 5 below:
| sample name | Peak area |
| Replicate-1 | 86.339 |
| Replicate-2 | 83.930 |
| Replicate-3 | 83.593 |
| Replicate-4 | 82.922 |
| Replicate-5 | 86.978 |
| Replicate-6 | 83.025 |
| Average value of | 84.464 |
| Relative standard deviation (%) | 1.892 |
The relative standard deviation was <2%, indicating good reproducibility of the method.
Example 4
(1) Sample preparation
An appropriate amount of ADC sample and CL2A-SN38 mother liquor were taken, acetonitrile was used: the aqueous solutions of water (25:75) were diluted into three sample solutions to be tested, sample 1-sample 3, respectively. Sample information is as follows:
| sample name | ADC concentration (mg/mL) | Addition amount of CL2A-SN38 (μM) |
| ADC blank sample | 1 | 0 |
| Labeled sample 1 | 1 | 2 |
| Labeling sample 2 | 1 | 5 |
(2) Chromatographic conditions:
mobile phase a was 0.1% aqueous trifluoroacetic acid and mobile phase B was 0.1% acetonitrile trifluoroacetic acid, elution gradients are as set forth in the following table:
| time (minutes) | Mobile phase A ratio (%) | Mobile phase B ratio (%) |
| 0 | 90 | 10 |
| 19 | 62.5 | 37.5 |
| 19.1 | 0 | 100 |
| 23 | 0 | 100 |
| 23.1 | 90 | 10 |
| 25 | 90 | 10 |
The chromatographic column used was advanced Bio RP-mAb Diphenyl (2.1X100 mm,3.5 μm). The wavelength of the ultraviolet detector is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 mu L, and the column temperature is 25 ℃.
(3) And (3) calculating results:
the CL2A-SN38 concentration of the triplicate samples was determined according to the linear equation obtained in example 3. The labeling recovery (%) = measured plus scalar/theoretical plus scalar x 100%. The spectrogram is shown in fig. 4, and the top down is respectively a blank solvent, an ADC blank sample, a labeled sample 1 and a labeled sample 2. According to the spectra, the ADC protein peak (FIG. 4: ADC peak) retention time was 20-22min, shortening the elution time compared to example 1. CL2A-SN38 has a peak (FIG. 4: drug peak) retention time of 15-17min, and good separation of small molecules and proteins. The retention time of the residual CL2A-SN38 correlation peak (figure 4:drug relatedpeak) in the ADC sample is respectively 5.5-6min,14-15min and 17.5-18min, and the sum of peak areas is recorded as the peak area of the CL2A-SN38 of the blank sample of the ADC.
The calculation results are shown in the following table:
the recovery rate is 80% -120%, and the labeling recovery rate of the method is good.
Comparative example 1
(1) Sample preparation
Taking a proper amount of ADC sample, adding a small amount of CL2A-SN38 mother liquor to obtain a mixed solution of ADC and CL2A-SN38, and adding 2 times volume of precipitant (10 g of NaCl is dissolved in a saturated solution of 30mL of mixed solution of methanol and 50mL of acetonitrile) to obtain a labeled sample 3. After the sample was sufficiently shaken for 10min, it was centrifuged at 16000rpm at room temperature for 10min, and 60. Mu.L of the supernatant was aspirated for sample introduction. Sample information is as follows:
(2) Chromatographic conditions:
mobile phase a was 0.1% aqueous trifluoroacetic acid and mobile phase B was 0.1% acetonitrile trifluoroacetic acid, elution gradients are as set forth in the following table:
| time (minutes) | Mobile phase A ratio (%) | Mobile phase B ratio (%) |
| 0 | 90 | 10 |
| 19 | 62.5 | 37.5 |
| 19.1 | 0 | 100 |
| 23 | 0 | 100 |
| 23.1 | 90 | 10 |
| 25 | 90 | 10 |
The chromatographic column used was advanced Bio RP-mAb Diphenyl (2.1X100 mm,3.5 μm). The wavelength of the ultraviolet detector is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 mu L, and the column temperature is 25 ℃.
(3) And (3) calculating results:
the CL2A-SN38 concentration of the sample was determined according to the linear equation obtained in example 3. The labeling recovery (%) = measured plus scalar/theoretical plus scalar x 100%. The spectrum is shown in FIG. 5, and according to the spectrum, CL2A-SN38 has a peak (FIG. 5: drug peak) retention time of 10-16.5min, and the main peak has a distinct front peak. The retention time of the residual CL2A-SN38 correlation peak (figure 5:drug relatedpeak) in the ADC sample is respectively 4-6min and 17.5-18min, and no obvious protein residue exists at the peak position of the protein. The calculation results are shown in the following table:
recovery was less than 80%, presumably the precipitation centrifugation step resulted in partial CL2A-SN38 co-precipitation, resulting in lower recovery.
Claims (10)
1. The liquid chromatography method for detecting the content of residual SN38 small molecules in the antibody coupled drug is characterized in that the method is characterized in that an ADC sample of the antibody coupled drug is directly injected or injected after dilution, and the content of free topoisomerase I inhibitor SN38 in the sample is detected by adopting a reversed phase chromatography method; the ADC sample is an antibody coupling drug coupled with SN 38; the mobile phase A of the reversed phase chromatography is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution; the elution gradient of the reverse phase chromatography is as follows:
。
2. The liquid chromatography method for detecting residual SN38 small molecule content in an antibody-conjugated drug according to claim 1, wherein the SN38 small molecule is a SN 38-based drug linker comprising CL2A-SN38, CL2E-SN38,
MC-VC-PAB-SN38。
3. the liquid chromatography method for detecting the residual SN38 small molecule content of an antibody-conjugated drug according to claim 1, wherein the diluent for ADC sample dilution is at least one of water, methanol, acetonitrile, ethanol, isopropanol.
4. The liquid chromatography method for detecting the residual SN38 small molecule content of an antibody-conjugated drug according to claim 3, wherein the diluent is a mixed solution of acetonitrile and water in a volume ratio of 25:75.
5. The liquid chromatography method for detecting the residual SN38 small molecule content of an antibody-conjugated drug according to claim 1, wherein the buffer of the ADC sample is at least one of histidine, arginine, sucrose, trehalose, and polysorbate.
6. The liquid chromatography for detecting the content of residual SN38 small molecules in an antibody-conjugated drug according to claim 1, wherein the chromatographic column of the reversed phase chromatography is a large-aperture reversed phase liquid chromatographic column suitable for protein separation, the wavelength of ultraviolet detection is 370nm, the flow rate is 0.5mL/min, the sample injection amount is 40 μl, and the column temperature is 25 ℃.
7. The liquid chromatography method for detecting the content of residual SN38 small molecules in an antibody-conjugated drug according to claim 1, wherein the method comprises the steps of drawing a linear standard curve of a SN38 small molecule standard, comprising the steps of:
(1) Dissolving an SN38 small molecular standard substance by using dimethylacetamide DMA to prepare a mother solution with the concentration of 10 mM;
(2) Preparing a mother solution prepared in the step (1) into a linear standard solution by adopting a diluent, wherein the concentration is as follows:
；
wherein the diluent is at least one of water, methanol, acetonitrile, ethanol and isopropanol.
8. The liquid chromatography for detecting residual SN38 small molecule content in an antibody-conjugated drug according to claim 7, wherein when diluted with a mixed solution of acetonitrile and water in a volume ratio of 50:50, a linear equation of a standard curve drawn with the concentration of the standard solution as the abscissa and the chromatographic peak area as the ordinate is: y= 72.677x-4.5826, r 2 = 0.9991; when the mixed solution of acetonitrile and water with the volume ratio of 25:75 is used for dilution, the linear equation of the drawn standard curve is as follows: y= 88.2245x-4.5635, r 2 ＝0.9991。
9. The liquid chromatography method for detecting the content of residual SN38 small molecules in an antibody-conjugated drug according to claim 1, wherein the specific steps of the method comprise:
s1, drawing a standard curve
Accurately weighing an SN38 small molecular standard substance, and dissolving by adopting dimethylacetamide DMA to prepare a mother solution; then gradually diluting with a diluent to prepare a linear standard solution, sampling and eluting, and preparing a linear standard curve by taking the concentration of the standard solution as an abscissa and the chromatographic peak area as an ordinate to obtain a linear equation;
s2, detecting SN38 content in antibody coupled drug ADC sample
And directly sampling or diluting an antibody-coupled drug ADC sample, sampling, eluting, reading chromatographic peak area, and calculating the concentration of SN38 in the sample and the labeling recovery rate according to the linear equation obtained in S1, wherein the labeling recovery rate/% = actual measurement plus scalar/theoretical plus scalar multiplied by 100%.
10. The liquid chromatography method for detecting the content of residual SN38 small molecules in an antibody-conjugated drug of claim 9, wherein the method has a labeling recovery of 80-120%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228508A (en) * | 2018-11-29 | 2020-06-05 | 暨南大学 | Multi-target anti-tumor polypeptide drug conjugate and preparation method and application thereof |
| CN111433188A (en) * | 2018-12-17 | 2020-07-17 | 荣昌生物制药(烟台)股份有限公司 | Linker for antibody drug conjugate and application thereof |
| CN112285238A (en) * | 2020-10-23 | 2021-01-29 | 东曜药业有限公司 | Liquid chromatography for detecting content of free micromolecules of ADC (azodicarbonamide) medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228508A (en) * | 2018-11-29 | 2020-06-05 | 暨南大学 | Multi-target anti-tumor polypeptide drug conjugate and preparation method and application thereof |
| CN111433188A (en) * | 2018-12-17 | 2020-07-17 | 荣昌生物制药(烟台)股份有限公司 | Linker for antibody drug conjugate and application thereof |
| CN112285238A (en) * | 2020-10-23 | 2021-01-29 | 东曜药业有限公司 | Liquid chromatography for detecting content of free micromolecules of ADC (azodicarbonamide) medicine |
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| Title |
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| RENU PANDEY, ALEKSANDRA GRUSLOVA, JENNIFER CHIOU, ET AL.: "Stable Isotope Dilution LC-HRMS Assay To Determine Free SN-38, Total SN-38, and SN-38G in a Tumor Xenograft Model after Intravenous Administration of Antibody-Drug Conjugate (Sacituzumab Govitecan)", 《ANALYTICAL CHEMISTRY》, vol. 92, no. 1, 7 January 2020 (2020-01-07), pages 1260 - 1267 * |
| ROBERT M. SHARKEY, SERENGULAM V. GOVINDAN, THOMAS M. CARDILLO, ET AL.: "Selective and Concentrated Accretion of SN-38 with a CEACAM5-Targeting Antibody-Drug Conjugate (ADC), Labetuzumab Govitecan (IMMU-130)", 《MOLECULAR CANCER THERAPEUTICS》, vol. 17, no. 1, 1 January 2018 (2018-01-01), pages 196 - 203, XP055856822, DOI: 10.1158/1535-7163.MCT-17-0442 * |
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