Method for detecting cysteine-conjugated ADC conjugation site distribution and occupancy, and method for calculating drug-antibody ratio

Abstract

The present invention provides a method for detecting the coupling site distribution and occupancy of cysteine-conjugated ADCs and a method for calculating the drug-antibody ratio. The detection method includes the following steps: treating the naked antibody and the ADC separately into peptide mixtures capable of LC-MS analysis; taking equal amounts of the peptide mixture of the naked antibody and the peptide mixture of the ADC for LC-MS analysis respectively; normalizing the responses of all peptides after LC-MS analysis using the responses of peptides without cysteine to obtain a normalization coefficient, and calculating the occupancy of the coupling sites of cysteine-conjugated ADCs based on the response values of cysteine-containing peptides corrected by the normalization coefficient. Through the above experimental operations, the present application avoids the quantitative errors caused by the large differences in the ionization efficiency of peptides due to drug modification, and solves the problem of low quantitative accuracy of the analysis site occupancy of current ADCs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis. Specifically, it relates to a method for detecting the distribution and occupancy of conjugation sites of cysteine-conjugated ADCs and a method for calculating the drug-antibody ratio. Background Art

[0002] Antibody-drug conjugates (ADCs) combine the specific recognition of monoclonal antibodies with the killing activity of small molecule cytotoxic drugs, improving the targeting of tumor drugs and reducing side effects. The accurate recognition of targets by ADC drugs greatly improves the drug efficacy and reduces side effects, attracting much attention from personnel in the pharmaceutical R & D field. The structure of ADC drugs is relatively complex, generally including antibodies, linkers, and small molecule drugs. The drug loading of small molecule drugs is crucial for the activity and toxicity of ADC drugs.

[0003] Currently, the calculation of the occupancy of conjugation sites of ADCs is a major difficulty in the analysis of antibody-drug conjugates. The traditional calculation of site occupancy is based on the percentage of the intensities of conjugated drug and non-conjugated drug peptides, and the calculation result has poor accuracy and is difficult to accurately quantify. Summary of the Invention

[0004] The main object of the present invention is to provide a method for detecting the distribution and occupancy of conjugation sites of cysteine-conjugated ADCs and a method for calculating the drug-antibody ratio, so as to solve the problem of low accuracy in detecting the occupancy of conjugation sites of ADCs in the prior art.

[0005] To achieve the above object, according to the first aspect of the present invention, a method for detecting the occupancy of conjugation sites of cysteine-conjugated ADCs is provided. The detection method includes the following steps: S1, processing the naked antibody and ADC into peptide mixtures capable of LC-MS analysis respectively; S2, taking equal amounts of the peptide mixture of the naked antibody and the peptide mixture of the ADC for LC-MS analysis respectively; S3, normalizing the responses of all peptides after LC-MS analysis using the response of the peptide without cysteine to obtain a normalization coefficient, and calculating the occupancy of the conjugation sites of the cysteine-conjugated drug based on the response value of the peptide containing cysteine corrected by the normalization coefficient.

[0006] Further, S3 includes: S31, calculating the normalization coefficient N of the responses of all peptides after LC-MS analysis using the response of the peptide without cysteine; S32, calculating the relative contents of the peptides containing cysteine in the naked antibody and the ADC after normalization using the normalization coefficient N for the same peptide containing cysteine in the ADC; S33, subtracting the relative content of the peptide containing cysteine in the ADC after normalization from 1.00 to obtain the occupancy of the corresponding conjugation site of the cysteine-conjugated drug.

[0007] Further, S31 includes: recording the response value of the cysteine - free peptide segment in the naked antibody as X, recording the response value of the cysteine - free peptide segment with the same sequence in the ADC as Y, and normalizing the response of the same cysteine - free peptide segment by X / Y; calculating the average value of the normalized responses of all cysteine - free peptide segments to obtain the normalization coefficient N.

[0008] Further, the cysteine - free peptide segments in the naked antibody and the cysteine - free peptide segments with the same sequence in the ADC are selected from the peptides of the top n sequences with the highest response values after LC - MS analysis, where n is a natural number between 3 and 10.

[0009] Further, S32 includes: recording the response value of the cysteine - containing peptide segment in the naked antibody as A, recording the response value of the cysteine - containing peptide segment with the same sequence in the ADC as B, and obtaining the content of the cysteine - containing peptide segment in the normalized ADC by (B / A)*N.

[0010] Further, S1 includes: sequentially performing a reduction reaction and an alkylation reaction on equal amounts of the naked antibody and the ADC to obtain an alkylated naked antibody and an alkylated ADC; performing enzymatic digestion on the alkylated naked antibody and the alkylated ADC to obtain a peptide mixture of the naked antibody and a peptide mixture of the ADC, respectively.

[0011] Further, the reduction reaction is carried out using a reducing agent; the reducing agent includes any one or more of tris(2 - carboxyethyl)phosphine, dithiothreitol, and β - mercaptoethanol.

[0012] Further, the alkylation reaction is carried out using an alkylating agent; the alkylating agent includes any one or more of N - ethylmaleimide, iodoacetamide, methyl methanesulfonate, and iodoacetic acid.

[0013] Further, the enzymatic digestion is carried out using a protease; the protease includes any one or more of endoprotease Arg - C, endoprotease Arg - N, endoprotease Lys - C, endoprotease Lys - N, endoprotease Asp - N, endoprotease Glu - C, and trypsin.

[0014] Further, before enzymatic digestion, the alkylated naked antibody and the alkylated ADC are filtered; the filtration includes: centrifugal filtration using an ultrafiltration tube; the pore size of the ultrafiltration tube is 3 - 30 kDa.

[0015] To achieve the above object, according to the second aspect of the present invention, a method for detecting the coupling site distribution of cysteine-conjugated ADC is provided. The detection method includes: detecting the occupancy of all coupling sites in the ADC by using the above method for detecting the occupancy of the coupling sites of cysteine-conjugated ADC; the occupancy of each peptide segment containing cysteine can indicate the position and proportion of the coupling site in the ADC sequence, constituting the distribution of the coupling sites of the ADC.

[0016] To achieve the above object, according to the third aspect of the present invention, a method for calculating the drug-antibody ratio of cysteine-conjugated ADC is provided. The calculation method includes: detecting the occupancy of all coupling sites in the ADC by using the above method for detecting the occupancy of the coupling sites of cysteine-conjugated ADC; adding up the occupancy of all coupling sites in the ADC, and the drug-antibody ratio of the cysteine-conjugated drug can be obtained.

[0017] Applying the technical solution of the present invention, in this application, the naked antibody and the ADC are processed in parallel, and the enzymatically cleaved peptide segments are identified by combining liquid chromatography-mass spectrometry (LC-MS). All peptide segments after LC-MS analysis are normalized by using peptide segments without cysteine to obtain the accurate relative content of peptide segments containing cysteine, and then the occupancy of each coupling site of cysteine-conjugated ADC is calculated. This experimental operation is simple, and the occupancy result is accurate, avoiding the quantitative error caused by the change in the ionization efficiency difference of peptide segments due to drug modification, and well solving the problem of low quantitative accuracy of the analysis site occupancy of ADC at present. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The specification drawings constituting a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0019] Figure 1 shows a schematic flow chart of the detection method according to the present invention;

[0020] Figure 2A shows the LC-MS BPC diagrams of the naked antibody and the ADC obtained in Example 2;

[0021] Figure 2B shows the extracted ion chromatograms of the coupling site peptide segments of the naked antibody and the ADC obtained in Example 2;

[0022] Figure 3A shows the LC-MS BPC diagrams of the naked antibody and the ADC obtained in Example 3;

[0023] Figure 3BShows the extracted ion chromatogram of the coupling site peptides of the naked antibody and ADC obtained in Example 3;

[0024] Figure 4A Shows the LC-MS BPC chromatograms of the naked antibody and ADC obtained in Example 4;

[0025] Figure 4B Shows the extracted ion chromatogram of the coupling site peptides of the naked antibody and ADC obtained in Example 4.

[0026] Figure 5A Shows the LC-MS BPC chromatograms of the naked antibody and ADC obtained in Example 5;

[0027] Figure 5B Shows the extracted ion chromatogram of the coupling site peptides of the naked antibody and ADC obtained in Example 5;

[0028] The above-mentioned Figure 2A 、 2B The "uncoupled protein" in 3A, 3B, 4A, 4B, 5A, 5B is a different expression of the same substance as the "naked antibody" in the specification of this application. Detailed implementation mode

[0029] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0030] As mentioned in the background art, antibody-drug conjugate (ADC), as a monoclonal antibody carrying a small molecule drug, can specifically recognize and kill cells. Therefore, the loading of the small molecule drug is crucial for both activity and toxicity. The traditional calculation method for the occupancy rate of the ADC coupling site only performs LC-MS analysis on the ADC, and the calculation formula is occupancy rate = response value of the ADC-coupled drug peptide / (response value of the ADC-coupled drug peptide + response value of the ADC non-coupled drug peptide). This calculation method cannot eliminate the errors caused by sample processing, ionization efficiency, and operation during the LC-MS analysis, and there is no reference control sample, so the accuracy of the calculated occupancy rate value is relatively poor. In order to reduce the calculation error of the occupancy rate of the ADC coupling site, based on the parallel processing of the naked antibody and ADC in this application, LC-MS analysis is used to normalize the response values of the cysteine alkylated peptides in the naked antibody and ADC, and the accurate calculation of the occupancy rate of the coupling site is completed.

[0031] In the first typical implementation mode of this application, a method for detecting the occupancy rate of the coupling site of a cysteine-coupled ADC is provided, and the method flow is as Figure 1As shown, the detection method includes the following steps: S1, processing the naked antibody (also called uncoupled protein in this application, with the two having exactly the same meaning) and the ADC into peptide mixtures capable of LC-MS analysis respectively; S2, taking equal amounts of the peptide mixture of the naked antibody and the peptide mixture of the ADC for LC-MS analysis respectively; S3, normalizing the responses of all peptides after LC-MS analysis using the responses of peptides without cysteine, obtaining a normalization coefficient, and correcting the response values of peptides containing cysteine based on the normalization coefficient, and then calculating the occupancy rate of the conjugation sites of the cysteine-conjugated drug.

[0032] Performing parallel sample processing on the naked antibody and the ADC before conventional LC-MS loading, and performing LC-MS analysis on the basis of ensuring equal amounts of the two samples. Normalizing the responses of all peptides after LC-MS analysis using the responses of peptides without cysteine, and calculating the occupancy rate of the conjugation sites of the ADC using the content of peptides containing cysteine after normalization processing can obtain more accurate results of the occupancy rate of the conjugation sites, providing strong data support for the therapeutic effect of the ADC drug.

[0033] In a preferred embodiment, the above S3 includes: S31, calculating the normalization coefficient N of the responses of all peptides after LC-MS analysis using the responses of peptides without cysteine; S32, calculating the relative contents of peptides containing cysteine in the naked antibody and the ADC after normalization processing using the normalization coefficient N based on the same peptide containing cysteine in the ADC; S33, subtracting the relative content of peptides containing cysteine in the ADC after normalization processing from 1.00 to obtain the occupancy rate of the corresponding conjugation sites of the cysteine-conjugated drug.

[0034] Using the responses of peptides without cysteine as the basic data for normalization, normalizing the response data of all peptides after LC-MS analysis using the obtained normalization coefficient, and calculating the relative content of peptides containing cysteine in the ADC after unifying the data to the same level can exclude the errors that may be caused to LC-MS analysis by sample pretreatment (inconsistent completion degrees of reduction, alkylation reactions, and enzymatic digestion of the two groups of samples of the naked antibody and the ADC, or inconsistent loss amounts during the reaction process of the two groups of samples, etc.) in the existing method for calculating the occupancy rate. Normalization eliminates the errors that may be generated between samples due to experimental processing operations to the greatest extent.

[0035] Among them, the content of the cysteine-containing peptide segment in the ADC represents the peptide segment content where the site without the conjugated small molecule drug is located in the ADC, and 1 minus the content of the cysteine-containing peptide segment after normalization in the ADC is the occupancy rate of the site conjugated with the small molecule drug in the ADC. In addition, the occupancy rate of the conjugation site mentioned in this application is the occupancy rate of each conjugation site. In the ADC conjugated with cysteine, there are 8 most common sites for conjugating small molecule drugs, namely the 4 cysteine sites of the two inter-chain disulfide bonds between the light chain and the heavy chain in the antibody, and the 4 cysteine sites of the two inter-chain disulfide bonds between the heavy chains. The sites and distribution of the conjugated drugs are key quality attributes for ADC drugs, so it is necessary to calculate indicators such as the occupancy rate of the conjugation sites.

[0036] In a preferred embodiment, the above S31 includes: recording the response value of the cysteine-free peptide segment in the naked antibody as X, recording the response value of the same sequence cysteine-free peptide segment in the ADC as Y, and using X / Y to normalize the response of the same cysteine-free peptide segment; calculating the average value of the normalized responses of all cysteine-free peptide segments to obtain the normalization coefficient N. In a preferred embodiment, the cysteine-free peptide segments in the naked antibody and the same sequence cysteine-free peptide segments in the ADC are selected from the top n peptide segments with the highest response value ratio after LC-MS analysis, and n is a natural number greater than or equal to 3; more preferably, n is a natural number between 3 and 15, and further preferably, n is a natural number between 3 and 10 (specifically, it can be 3, 4, 5, 6, 7, 8, 9, 10).

[0037] Sorting the response value intensities of all peptide segments obtained by LC-MS analysis of the naked antibody and the ADC, and selecting the peptide segments with higher response intensities for subsequent normalization analysis can ensure the accuracy of the basic response data for normalization, and minimize the possible errors in the normalization data processing process. In the embodiments of this application, the top 8 peptide segments with the highest response intensities are selected to determine the normalization coefficient. In addition, selecting the average value of the normalization coefficients calculated from multiple peptide segments can further ensure the accuracy of the response values in this normalization process.

[0038] In a preferred embodiment, the above S32 includes: recording the response value of the cysteine-containing peptide segment in the naked antibody as A, recording the response value of the same sequence cysteine-containing peptide segment in the ADC as B, and using (B / A)*N to obtain the content of the cysteine-containing peptide segment in the ADC after normalization.

[0039] Normalize the response of the naked antibody and the cysteine-containing peptide segments in the ADC with a normalization coefficient, where the cysteine-containing peptide segments in the naked antibody are all the peptide segments on the antibody that can be conjugated with small molecule drugs, and the cysteine-containing peptide segments in the ADC for which a response value can be detected belong to the peptide segments at the sites where small molecule drugs are not conjugated. Therefore, (B / A)*N gives the relative content of the cysteine-containing peptide segments in the naked antibody and the ADC, and also represents the occupancy of the sites in the ADC where small molecule drugs are not conjugated.

[0040] In a preferred embodiment, the above S1 includes: performing a reduction reaction and an alkylation reaction on an equal amount of the naked antibody and the ADC in sequence to obtain an alkylated naked antibody and an alkylated ADC; performing enzymatic digestion on the alkylated naked antibody and the alkylated ADC to obtain a peptide segment mixture of the naked antibody and a peptide segment mixture of the ADC. In a preferred embodiment, a reducing agent is used for the reduction reaction. Any reducing agent capable of completing the reduction reaction is applicable to this application. In a preferred embodiment, the reducing agent includes any one or more of tris(2-carboxyethyl)phosphine, dithiothreitol, and β-mercaptoethanol.

[0041] Perform pretreatment on the naked antibody and ADC samples before LC-MS injection, including reducing the samples to open the interchain disulfide bonds present in the antibody and reduce them to cysteine, and then performing an alkylation reaction on the reduced samples to prevent the reformation of disulfide bonds. Completing the enzymatic digestion of the alkylated samples completes the pretreatment before LC-MS injection. Among them, in order to minimize the errors that may be caused to the LC-MS analysis results in the experimental operations of the pretreatment, quantitative control of equal sampling of the naked antibody and ADC samples was carried out at the beginning of sampling.

[0042] In a preferred embodiment, an alkylating agent is used for the alkylation reaction; any alkylating agent capable of completing the alkylation reaction is applicable to this application. In a preferred embodiment, the alkylating agent includes any one or more of N-ethylmaleimide, iodoacetamide, methyl methanesulfonate, and iodoacetic acid.

[0043] In a preferred embodiment, a protease is used for enzymatic digestion. Any protease capable of digesting the samples to obtain a peptide segment mixture is applicable to this application. In a preferred embodiment, the protease includes any one or more of endoprotease Arg-C, endoprotease Lys-C, endoprotease Arg-N, endoprotease Lys-N, endoprotease Asp-N, endoprotease Glu-C, and trypsin. Among them, endoprotease Arg-C is a protease that specifically cleaves the carboxyl terminus of arginine; endoprotease Lys-C is a protease that specifically cleaves the carboxyl terminus of lysine.

[0044] In a preferred embodiment, before enzymatic hydrolysis, the alkylated naked antibody and alkylated ADC are filtered; any method capable of filtering and removing impurities from the reaction product is applicable to this application. In a preferred embodiment, the filtration includes centrifugal filtration using an ultrafiltration tube. In a preferred embodiment, the pore size of the ultrafiltration tube is 3 - 30 Kd. Filtering the product after the alkylation reaction and performing enzymatic hydrolysis after impurity removal can obtain relatively accurate result data for LC-MS analysis in this operation.

[0045] In the second typical embodiment of this application, a method for detecting the coupling site distribution of cysteine-conjugated ADC is provided. The detection method includes: detecting the occupancy rate of all coupling sites in the ADC using the above-mentioned method for detecting the occupancy rate of the coupling sites of cysteine-conjugated ADC; the positions and proportions of the cysteine-containing peptide segments with each occupancy rate in the ADC structure constitute the distribution of each coupling site. By analyzing the peptide segments at the sites with occupancy rates, the distribution of the coupling sites can be known. The occupancy rates obtained in this application are relatively accurate, so the specific distribution information of each coupling site obtained is also more accurate, providing a reliable understanding of the distribution of each site of the ADC drug and providing data support for subsequent administration.

[0046] In the third typical embodiment of this application, a method for calculating the drug-antibody ratio of cysteine-conjugated ADC is provided. The calculation method includes: detecting the occupancy rate of all coupling sites in the ADC using the above-mentioned method for detecting the occupancy rate of the coupling sites of cysteine-conjugated ADC; adding up the occupancy rates of all coupling sites in the ADC, and the drug-antibody ratio of the cysteine-conjugated drug is obtained. The drug-antibody ratio (DAR) represents the average number of small molecule drugs conjugated to the antibody. By adding up the occupancy rates of the coupling sites where small molecule drugs exist, the DAR of this ADC can be obtained. Since the occupancy rates of the coupling sites obtained in this application are relatively accurate, the DAR value obtained thereby is also more credible. Compared with the existing DAR value characterization and analysis methods, such as HIC-HPLC, which can separate and purify ADC under natural conditions and obtain the corresponding DAR value, but this method cannot be compatible with MS analysis and the experimental operation is relatively complex.

[0047] The following further describes this application in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed by this application.

[0048] Example 1: A method for analyzing the coupling site distribution and occupancy rate of cysteine-conjugated ADC

[0049] The steps are as follows:

[0050] (1) Pipette 200 μg of the naked antibody sample and the ADC sample into separate 1.5 mL centrifuge tubes, add 0.1 M Tris-HCl buffer containing 8 M guanidine hydrochloride (Gdn·HCl) to 200 μL, mix well, and dilute the samples to 1 mg / mL.

[0051] (2) Add 8 μL of 0.5 M TCEP (tris(2-carboxyethyl)phosphine) to each, with a total system volume of 108 μL. After mixing, incubate in a 56 °C water bath or metal bath for 30 ± 5 min.

[0052] (3) Add 20 μL of 0.5 M IAM (iodoacetamide) to the samples and incubate in the dark at room temperature for 30 ± 5 min.

[0053] (4) Transfer the sample solution to a 10 KD ultrafiltration tube, add 400 μL of 0.1 M Tris-HCl, and centrifuge at 13,000 rcf at 10 °C for 15 - 20 min. Discard the waste liquid in the outer tube and repeat this step once.

[0054] (5) Invert the inner core of the ultrafiltration tube into a new outer jacket and centrifuge briefly to recover the protein. Transfer the recovered sample solution to a new 1.5 mL low-protein-binding centrifuge tube. After measuring the sample concentration, pipette 100 μg of the protein sample, add 0.1 M Tris-HCl (pH 7.6) to make up the volume to 100 μL. Add LysC enzyme to the sample so that the mass ratio of the sample to the enzyme is 20:1. Mix well and digest at 37 °C for 1 h ± 10 min.

[0055] (6) Take out the sample and cool it to room temperature. Add 1 μL of 10% FA to terminate the reaction, centrifuge briefly at 13,000 rcf, transfer the supernatant to a liquid-phase vial, and perform LC-MS analysis. The injection volume and analysis method should be kept consistent.

[0056] (7) The mobile phase A for LC-MS is an aqueous solution containing 0.05 - 0.1% FA / 0 - 0.05% TFA, and the mobile phase B is an acetonitrile solution containing 0.05 - 0.1% FA / 0 - 0.05% TFA. Phase B is 0.5% B from 0 - 1 minute; from 1 - 80 minutes, 0.5 - 25% B is used respectively; from 80 - 95 minutes, 25 - 37% B is used respectively; then from 105 - 106 minutes, 37 - 50% B is used, from 106 - 111 minutes, isocratic elution with 90% B is used, from 111 - 112 minutes, 90 - 0.5% B is used, and from 112 - 120 minutes, 0.5% B is used for equilibration.

[0057] (8) Calculate the response ratios of the 8 peptides with the highest response intensity that do not contain cysteine in the naked antibody and the ADC respectively. Let the response of the peptide in the naked antibody be X and the response of the corresponding peptide in the ADC be Y. If there is an offset, normalization is required to calculate the normalization coefficient N (N = Average(X / Y)).

[0058] (9) Calculate the ratios of the peptides containing cysteine in the naked antibody and the ADC respectively. Let the response of the peptide in the naked antibody be A and the response of the corresponding cysteine-containing peptide in the ADC be B. Calculate the occupancy rate of the corresponding site in the ADC: Occupancy rate = (1 - (B / A) * N), which is the occupancy rate of the corresponding site. From the occupancy rate information, the distribution of the drug and DAR (Sum(occupancy rate * number of sites with the same occupancy rate)) in the ADC can be obtained.

[0059] Example 2

[0060] The sample is a cysteine-conjugated ADC synthesized in the laboratory. The drug-linker used in Examples 2 and 3 is vc-MMAE, which is synthesized in the laboratory. The protein is a commercial monoclonal antibody Herceptin (a product of Roche). The difference is that Example 2 is a high-DAR ADC synthesized in the laboratory, while Example 3 is a low-DAR ADC synthesized in the laboratory. These two examples are used to prove that the method is not affected by DAR changes and has wide adaptability.

[0061] The DAR value of Example 2 was determined to be 4.5 by HIC (hydrophobic interaction chromatography, a method commonly used for ADC DAR value analysis). After measuring the protein concentration by UV280, the coupling site distribution and occupancy rate of the ADC synthesized in the laboratory were analyzed according to the method of Example 1.

[0062] After matching the mass spectrometry spectra, 8 peptides with the highest response intensity that do not contain cysteine peptides in the naked antibody and the ADC were screened. The normalization factor N was calculated by the responses of the Top 8 peptides in the naked antibody and the conjugated ADC. As shown in Table 1, the normalization factor N of this sample is 2.01. By quantifying the responses of the peptides containing cysteine in the protein before conjugation and the ADC after conjugation, it can be obtained that the ADC conjugation sites involve a total of 8 sites, namely the four cysteines connected by the light and heavy chains and the four cysteines connected between the heavy chains.

[0063] Among them, there are 2 cysteine sites at the light chain terminus, corresponding to the peptide segment SFNRGEC (SEQ ID NO:1), and the average cysteine occupancy is 0.58. There are 2 cysteine sites at the junction of the heavy chain and the light chain, corresponding to the peptide segment SCDK (SEQ ID NO:2), and the average cysteine occupancy is 0.71. There are 4 cysteine sites at the junction between the heavy chains, corresponding to the peptide segment THTCPPCPAPELLGGPSVFLFPPK (SEQ ID NO:3), and the average occupancy of the two cysteine sites is 0.44. (Note: For the case where a peptide segment has two cysteines, if a more accurate distribution is required, it can be based on one cysteine modified conjugate and one cysteine modified alkylating reagent. In this experiment, it is defaulted that the two sites are equally occupied by the conjugate.)

[0064] In summary, the conjugation sites and site occupancy information of the ADC can be obtained, as shown in Table 2. The DAR value calculated by this method is 3.5, slightly lower than the HIC result. According to subsequent analysis, the proportion of the two cysteines in the hinge region of this molecule that are conjugated is relatively high. If the occupancy of each cysteine site is 0.44, the DAR value calculated by this method is 4.4, which is basically consistent with the HIC result.

[0065] The LC-MS BPC of the naked antibody and the conjugated ADC obtained in this example, and the extracted ion chromatograms of the conjugated site peptide segments are as Figure 2A and Figure 2B shown. Among them, Figure 2A is the base peak chromatogram (BPC chromatogram), which is a chromatogram obtained by continuously depicting the intensity of the strongest ion in the mass spectrum at each time point. Each peak in the chromatogram represents the elution of the peptide segment at the time point, proving that the peptide segment has good chromatographic separation throughout the gradient range, while Figure 2B the b, c, and d graphs in are the extracted ion chromatograms of the peptide segments SCDK, peptide segment SFNRGE C, and peptide segment THTCPPCPAPELLGGPSVFLFPPK respectively. Among them, the peak represents the response intensity of the peptide segment, and the corresponding peak area is the response value (A, B).

[0066] Table 1 Calculation of the normalization coefficient N

[0067]

[0068] Table 2 Calculation of site occupancy and DAR value

[0069]

[0070] Example 3

[0071] The sample is an ADC synthesized in the laboratory. After measuring the protein concentration by UV280, 200 μg of the protein before conjugation and the ADC after conjugation were taken respectively. After denaturation, reduction alkylation, buffer exchange, Lys-C / Trypsin digestion, the protein concentration of the digested sample was measured again by UV280, and an equal amount of the sample was taken for LC-MS / MS analysis. The DAR value of Example 3 was determined to be 3.5 by HIC (hydrophobic interaction chromatography, a method commonly used for ADC DAR value analysis).

[0072] After matching the mass spectrometry spectra by software and manually, 8 peptides with the highest response intensity of the naked antibody and the cysteine-free peptides in the ADC were screened. The normalization factor N was calculated by the responses of the Top 8 peptides in the naked antibody and the conjugated ADC. As shown in Table 3, the normalization factor N of this sample was 0.95. By quantifying the responses of the peptides containing cysteine in the protein before conjugation and the ADC after conjugation, it was found that there were 8 conjugation sites involved in this ADC, namely the four cysteines at the light and heavy chain junctions and the four cysteines at the heavy chain junctions respectively.

[0073] Among them, there were 2 cysteine sites at the light chain terminal, corresponding to the peptide GEC, and the average cysteine occupancy was 0.44. There were 2 cysteine sites at the light and heavy chain junctions, corresponding to the peptide SCDK, and the average cysteine occupancy was 0.81. There were 4 cysteine sites at the heavy chain junctions, corresponding to the peptide THTCPPCPAPELLGGPSVFLFPPK, and the average site occupancy of the two cysteines was 0.39. (Note: For the case where a peptide has two cysteines, if a more accurate distribution is required, one cysteine can be modified with a conjugate and the other cysteine can be modified with an alkylating reagent. In this experiment, it is defaulted that the two sites are equally occupied by the conjugate.)

[0074] In summary, the conjugation sites and site occupancy information of the ADC can be obtained, as shown in Table 4. The DAR value calculated by this method was 3.3, and this result was basically consistent with other DAR measurement results. Further analysis found that for low-DAR value molecules, the situation of two cysteine conjugations occurring simultaneously in the hinge region was less, so the data was more accurate when simplifying the calculation.

[0075] The LC-MS BPC of the naked antibody and the conjugated protein obtained in this example and the extracted ion chromatogram of the conjugated site peptides are shown in Figure 3A and Figure 3B as follows. Among them, Figure 3A is the BPC map, which is a map obtained by continuously depicting the intensity of the strongest ions in the mass spectrometry spectra at each time point. Each peak in the map represents the elution of peptides at the time point, proving that the peptides have good chromatographic separation throughout the gradient range; while Figure 3BThe extraction ion chromatograms of peptide segments SCDK, GEC, and THTCPPCPAPELLGGPSVFLFPPK are shown in Figures b, c, and d respectively. Among them, the peak represents the response intensity of the peptide segment, and the corresponding peak area is the response value (A, B).

[0076] Table 3 Calculation of the normalization coefficient N

[0077]

[0078] Table 4 Calculation of site occupancy and DAR value

[0079]

[0080] Example 4

[0081] The sample is a cysteine-conjugated ADC synthesized in the laboratory. The drug-linker used is vc-MMAE, and the protein is a monoclonal antibody against CD19 (NCBI ID: 930). After measuring the protein concentration by UV280, 400 μg of the protein before conjugation and the ADC after conjugation were each taken, denatured, reduced, alkylated with IAM, buffer exchanged, digested with Lys-C, and the protein concentration of the digested sample was measured again by UV280. Equal amounts of the samples were taken for LC-MS / MS analysis. The DAR value of Example 4 was determined to be 3.9 by HIC.

[0082] After matching the mass spectrometry spectra by software and manually, 8 peptide segments with the highest response intensities in the naked antibody and the heavy and light chains of the ADC that do not contain cysteine peptide segments were screened respectively. The normalization factors N of the heavy and light chains were calculated based on the responses of the Top 8 peptide segments in the naked antibody and the ADC after conjugation, as shown in Table 5. The normalization factor N of this sample is 1.09. By quantifying the responses of the peptide segments containing cysteine in the protein before conjugation and the ADC after conjugation, it can be obtained that there are a total of 8 sites involved in the conjugation of this ADC, namely the four cysteines at the connection of the heavy and light chains and the four cysteines at the connection between the heavy chains.

[0083] Among them, there are 2 cysteine sites at the light chain terminal, corresponding to the peptide segment SFNRGEC, and the average cysteine occupancy is 0.61. There are 2 cysteine sites at the connection between the heavy chain and the light chain, corresponding to the peptide segment SCDK, and the average cysteine occupancy is 0.55. There are 4 cysteine sites at the connection between the heavy chains, corresponding to the peptide segment THTCPPCPAPELLGGPSVFLFPPK, and the average site occupancy of the two cysteines is 0.50 each. (Note: For the case where a peptide segment has two cysteines, if a more accurate distribution is required, one cysteine can be modified with a conjugate and the other cysteine can be modified with an alkylating reagent. In this experiment, it is defaulted that the two sites are equally occupied by the conjugate.)

[0084] In summary, the conjugation sites and site occupancy information of the ADC can be obtained, as shown in Table 6. The DAR value calculated by this method is 3.3.

[0085] The LC-MS BPC of the naked antibody and the conjugated ADC, and the extracted ion chromatograms of the conjugated site peptides obtained in this example are as Figure 4A and Figure 4B shown. Among them, Figure 4A is the BPC map, which is obtained by continuously depicting the intensities of the strongest ions in the mass spectra at each time point. Each peak in the figure represents the elution of the peptide at the time point, demonstrating good chromatographic separation of the peptide throughout the gradient range. And Figure 4B Figures b, c, and d in it are the extracted ion chromatograms of peptide SCDK, peptide SFNRGEC, and peptide THTCPPCPAPELLGGPSVFLFPPK, respectively. Among them, the peak represents the response intensity of the peptide, and the corresponding peak area is the response value (A, B).

[0086] Table 5 Calculation of normalization coefficient N

[0087]

[0088] Table 6 Calculation of site occupancy and DAR value

[0089]

[0090] Example 5

[0091] This sample is a cysteine-conjugated ADC synthesized in the laboratory. The drug-linker used is vc-MMAE, and the protein is a CD19 antibody. After measuring the protein concentration by UV280, 400 μg of the protein before conjugation and the ADC after conjugation were each taken, denatured, reduced, alkylated with NEM, buffer exchanged, digested with Lys-C enzyme, and the protein concentration of the digested sample was measured again by UV280. Equal amounts of the samples were taken for LC-MS / MS analysis. The DAR value of Example 6 was determined to be 3.9 by HIC.

[0092] After matching the mass spectra by software and manually, 8 peptides with the highest response intensities in the naked antibody and the heavy and light chains of the ADC that do not contain cysteine peptides were screened respectively. The normalization factors N of the heavy and light chains were calculated by the responses of the Top 8 peptides in the naked antibody and the conjugated ADC, as shown in Table 7. The normalization factor N of this sample is 1.16. By quantitatively analyzing the responses of the peptides containing cysteine in the protein before conjugation and the ADC after conjugation, it can be obtained that the conjugation sites of this ADC involve a total of 8 sites, namely the four cysteines connecting the heavy and light chains and the four cysteines connecting between the heavy chains.

[0093] Among them, there are 2 cysteine sites at the light chain terminus, corresponding to the peptide segment SFNRGEC, with an average cysteine occupancy of 0.61. There are 2 cysteine sites at the junction of the heavy chain and the light chain, corresponding to the peptide segment SCDK, with an average cysteine occupancy of 0.85. There are 4 cysteine sites connecting the heavy chains to each other, corresponding to the peptide segment THTCPPCPAPELLGGPSVFLFPPK, and the average site occupancy of the two cysteines is 0.63 each. (Note: For the case where a peptide segment has two cysteines, if a more accurate distribution is required, it can be based on one cysteine modified conjugate and one cysteine modified alkylating reagent. In this experiment, it is defaulted that the two sites are equally occupied by the conjugate.)

[0094] In summary, the conjugation sites and site occupancy information of the ADC can be obtained as shown in Table 8, and the DAR value calculated by this method is 4.2.

[0095] The LC-MS BPC of the naked antibody and the conjugated ADC obtained in this example and the extracted ion chromatograms of the conjugated site peptide segments are as Figure 5A and Figure 5B shown. Among them, Figure 5A is the BPC graph, which is a graph obtained by continuously depicting the intensity of the strongest ion in the mass spectrometry graph at each time point. Each peak in the graph represents the elution of the peptide segment at the time point, proving that the peptide segment has good chromatographic separation throughout the gradient range, while Figure 5B Figures b, c, and d in it are the extracted ion chromatograms of the peptide segments SCDK, SFNRGEC, and THTCPPCPAPELLGGPSVFLFPPK respectively. Among them, the peak represents the response intensity of the peptide segment, and the corresponding peak area is the response value (A, B).

[0096] Table 7 Calculation of the normalization coefficient N

[0097]

[0098] Table 8 Calculation of site occupancy and DAR value

[0099]

[0100] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: In this application, the naked antibody and ADC samples are processed in parallel, and after LC-MS analysis, the response of all peptide segments is normalized using the response of the cysteine-free peptide segments. Then, the occupancy rate of the conjugation site is calculated using the response value of the cysteine-containing peptide segments after normalization, and the distribution of the conjugation sites is further obtained. The experimental operation for the occupancy rate of the ADC conjugation site in this application is simple, and the occupancy rate result is accurate. It avoids the quantitative error caused by the large difference in the ionization efficiency of peptide segments due to drug modification, and well solves the problem of low quantitative accuracy of the analysis site occupancy rate of current ADCs. At the same time as calculating the occupancy rate of the conjugation site, information on the conjugation site distribution and DAR can also be obtained. In addition, the method of this application is not affected by the Drug and Drug-linker, and is applicable to all ADCs conjugated with cysteine.

[0101] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, 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 occupancy rate of the conjugation site of a cysteine - conjugated ADC, characterized in that, the detection method comprises the following steps: S1, processing the naked antibody and the ADC into peptide mixtures capable of LC - MS analysis respectively; S2, taking equal amounts of the peptide mixture of the naked antibody and the peptide mixture of the ADC for LC - MS analysis respectively; S3, normalizing the responses of all peptides after LC - MS analysis using the response of the peptide without cysteine, obtaining a normalization coefficient, and calculating the occupancy rate of the conjugation site of the cysteine - conjugated ADC based on the response value of the peptide containing cysteine corrected by the normalization coefficient; S3 includes: S31, calculating the normalization coefficient N of the responses of all peptides after the LC - MS analysis using the response of the peptide without cysteine; S32, calculating the relative contents of the peptides containing cysteine in the naked antibody and the ADC after normalization using the normalization coefficient N for the same peptide containing cysteine in the ADC; S33, subtracting the relative content of the peptide containing cysteine in the ADC after normalization from 1.00 to obtain the occupancy rate of the corresponding conjugation site of the cysteine - conjugated ADC; S31 includes: Denoting the response value of the peptide without cysteine in the naked antibody as X, and the response value of the peptide without cysteine with the same sequence in the ADC as Y, and normalizing the response of the same peptide without cysteine using X / Y; Calculating the average value of the normalization of the responses of all peptides without cysteine to obtain the normalization coefficient N; S32 includes: Denoting the response value of the peptide containing cysteine in the naked antibody as A, and the response value of the peptide containing cysteine with the same sequence in the ADC as B, and obtaining the relative content of the peptide containing cysteine in the ADC after normalization using (B / A)*N.

2. The detection method according to claim 1, characterized in that, the peptides without cysteine in the naked antibody and the peptides without cysteine with the same sequence in the ADC are selected from the peptides of the top n sequences with the highest response value ratio after the LC - MS analysis, and n is a natural number between 3 and 10.

3. The detection method according to claim 1 or 2, characterized in that, S1 includes: Successively performing a reduction reaction and an alkylation reaction on equal amounts of the naked antibody and the ADC to obtain an alkylated naked antibody and an alkylated ADC; Performing enzymatic hydrolysis on the alkylated naked antibody and the alkylated ADC to obtain the peptide mixture of the naked antibody and the peptide mixture of the ADC respectively.

4. The detection method according to claim 3, characterized in that, using a reducing agent for the reduction reaction; the reducing agent includes any one or more of tris(2 - carboxyethyl)phosphine, dithiothreitol, and β - mercaptoethanol.

5. The detection method according to claim 3, characterized in that, using an alkylating agent for the alkylation reaction; The alkylating agent includes any one or more of N-ethylmaleimide, iodoacetamide, methyl methanesulfonate, and iodoacetic acid.

6. The detection method according to claim 3, characterized in that the enzymatic hydrolysis is carried out using a protease, and the protease includes any one or more of endoprotease Arg-C, endoprotease Arg-N, endoprotease Lys-C, endoprotease Lys-N, endoprotease Asp-N, endoprotease Glu-C, and trypsin.

7. The detection method according to claim 3, characterized in that before the enzymatic hydrolysis, the alkylated naked antibody or the alkylated ADC is filtered; the filtration includes: centrifugal filtration using an ultrafiltration tube; the pore size of the ultrafiltration tube is 3 - 30 kDa.

8. A detection method for the coupling site distribution of a cysteine-conjugated ADC, characterized in that the detection method includes: detecting the occupancy of all coupling sites in the ADC by using the detection method for the occupancy of the coupling sites of the cysteine-conjugated ADC according to any one of claims 1 to 7; According to the occupancy of each cysteine-containing peptide segment, the position and proportion of each coupling site in the ADC sequence can be known, and further the coupling site distribution of the cysteine-conjugated ADC can be known.

9. A calculation method for the drug-antibody ratio of a cysteine-conjugated ADC, characterized in that the calculation method includes: detecting the occupancy of all coupling sites in the ADC by using the detection method for the occupancy of the coupling sites of the cysteine-conjugated ADC according to any one of claims 1 to 7; performing a summation calculation on the occupancy of all coupling sites in the ADC, and the drug-antibody ratio of the cysteine-conjugated ADC is obtained.

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