Liquid chromatography method for simultaneously detecting ADC drug antibody coupling ratio and free small molecule content and application thereof

Abstract

The invention provides a liquid chromatography method for simultaneously detecting ADC drug antibody coupling ratio and free small molecule content and application thereof, the method comprises the following steps: detecting and analyzing an ADC drug sample by hydrophobic interaction chromatography, the wavelength collection range of a detector in the chromatographic condition comprises 270nm-290nm and 340nm-390nm; the drug antibody coupling ratio is calculated based on data of the wavelength of 270-290 nm, and peaks belong to an uncoupled antibody peak, a D2 peak, a D4 peak, a D6 peak or a D8 peak; the proportion of each peak area reflects proportion distribution of different coupling ratios, and carrying out weighted calculation on the peak areas to obtain an average drug antibody coupling ratio; and calculating the content of the free small molecules based on the data of the wavelength of 340-390nm, assigning the peak to a coupled antibody peak and a free small molecule peak, and calculating the ratio of the peak area of the free small molecules to the total peak area to obtain the content of the free small molecules.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a liquid chromatography method for simultaneously detecting an ADC drug-antibody coupling ratio and a free small molecule content, and an application thereof. Background Art

[0002] ADC drugs use specific linkers to connect antibodies and small molecule cytotoxic drugs. Its main components include antibodies, linkers and small molecule cytotoxic drugs. Generally speaking, antibody molecules mainly play a targeted delivery role, and small molecule drugs play an effective role. However, some antibodies also have anti-tumor pharmacodynamic effects.

[0003] The drug-antibody coupling ratio and free small molecule content of ADC products are their key quality attributes, which have an important impact on drug efficacy and safety. In the existing technology, different chromatographic systems are often required for separate detection, such as using hydrophobic interaction chromatography to detect the drug-antibody coupling ratio and using reversed-phase liquid chromatography to determine the free small molecule content of ADC.

[0004] Since free small molecules can be dissolved in organic solvents, the content of free small molecules can be determined by reversed-phase liquid chromatography. In order to avoid the retention of large molecular proteins on the reversed-phase chromatographic column, sample pretreatment is usually required. First, the ADC sample is treated with a protein precipitant, and then the protein is removed by centrifugation. Finally, the supernatant containing small molecules is quantitatively analyzed. The disadvantages are: 1. The types of protein precipitants need to be screened, and the conditions of centrifugation need to be explored, which is time-consuming; 2. The pretreatment steps are cumbersome, and protein residues are prone to affect quantitative detection. The above method detects the drug-antibody coupling ratio and free small molecule content based on different chromatographic systems, and the process is complicated and the detection cost is high.

[0005] Therefore, developing a method that can simultaneously detect the antibody coupling ratio and free small molecule content has important application value for the development of ADC products. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a liquid chromatography method and its application for simultaneously detecting the drug-antibody coupling ratio and free small molecule content of ADC samples. The present invention adopts a chromatographic method, which can simultaneously detect the drug-antibody coupling ratio, drug loading distribution and free small molecule content of ADC samples, overcomes the shortcomings of the prior art, simplifies the sample analysis process, and provides an analysis method that can simultaneously detect multiple key quality attributes of antibody-drug conjugates.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content, the method comprising: using hydrophobic interaction chromatography to detect and analyze the ADC drug sample, wherein the detector acquisition wavelength range in the chromatographic conditions includes 270nm-290nm and 340nm-390nm;

[0009] The drug-antibody coupling ratio is calculated based on the data of 270nm-290nm wavelength, and the peaks are classified as uncoupled antibody peaks, D2 peaks, D4 peaks, D6 peaks or D8 peaks; the proportion of each peak area reflects the distribution of different coupling ratios, and the average drug-antibody coupling ratio is obtained by weighted calculation of the peak area;

[0010] The free small molecule content was calculated based on the data at a wavelength of 340nm-390nm, the peaks were attributed to the coupled antibody peaks and the free small molecule peaks, and the ratio of the peak area of ​​the free small molecule to the total peak area was calculated to obtain the free small molecule content.

[0011] In the present invention, the free small molecule content refers to the molar ratio of free small molecules in the ADC stock solution or finished product to all small molecules in the ADC sample.

[0012] The method of the present invention is to use direct injection method to perform liquid chromatography analysis on ADC samples. Based on the direct injection method of hydrophobic interaction chromatography, the drug-antibody coupling ratio, drug loading distribution and free small molecule content of the sample are detected simultaneously. The advantages are:

[0013] (1) The method can simultaneously report the drug-antibody coupling ratio, drug loading distribution and free small molecule content of the ADC sample in one experiment, greatly shortening the detection time;

[0014] (2) Compared with the existing reported method (a liquid chromatography method for detecting the free small molecule content of ADC drugs), which requires the complex operation of protein precipitation and removal using an organic solvent salt solution, the sample can be directly injected without the need for complex operations such as precipitation and centrifugation, thus avoiding the impact of complex pretreatment on the sample;

[0015] (3) The free small molecule content can be directly reported by peak area ratio, without the need to use a more complicated external standard method for detection, which significantly reduces data processing time and improves detection efficiency.

[0016] Preferably, the small molecule type in the ADC is a Dxd type small molecule.

[0017] The Dxd small molecules of the present invention refer to exotecan and its derivatives.

[0018] The small molecule type on the ADC drug in the present invention is a Dxd type small molecule, and the UV absorption spectra of naked antibody and ADC are as follows: Figure 1As shown, since the ultraviolet absorption intensity of ADC and naked antibody in the wavelength range of 1: 270nm-290nm is high, the data in this wavelength range can be collected and processed to obtain the drug loading distribution, and then calculate its average drug-antibody coupling ratio; since the naked antibody has no ultraviolet absorption in the wavelength range of 2: 340-390nm, the small molecules contribute to all the ultraviolet absorption of the ADC sample in this wavelength range, so the data at this wavelength can be collected and processed to obtain the free small molecule content.

[0019] The ADC drug sample of the present invention can be directly injected for detection, without the need for protein precipitation and external standard methods to detect the content of free small molecules in the sample, and simultaneously detect the drug-antibody coupling ratio and drug loading distribution of the sample.

[0020] Preferably, the mobile phase in the chromatographic conditions comprises:

[0021] Mobile phase A: a mixed aqueous solution of 1.4-1.6 M ammonium sulfate and 47-53 mM potassium dihydrogen phosphate, pH 7.0 ± 0.3;

[0022] Mobile phase B: 47-53 mM potassium phosphate dibasic in water containing 10-30% isopropanol.

[0023] Among them, 1.4-1.6M ammonium sulfate can be, for example, 1.4M, 1.5M or 1.6M, etc.; 47-53mM dipotassium hydrogen phosphate can be, for example, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM or 53mM, etc.; pH 7.0±0.3 can be, for example, 6.7, 7.0 or 7.3, etc.; 10-30% isopropanol can be, for example, 10%, 15%, 20%, 25% or 30%, etc.

[0024] Preferably, the elution procedure in the chromatographic conditions comprises:

[0025] In the first 0-1 minute, the volume fraction of mobile phase A was 98-100%, and the rest was mobile phase B;

[0026] In the first 3 minutes, the volume fraction of mobile phase A changes from 98-100% to 68-70% at a constant rate, and the rest is mobile phase B;

[0027] From 3 to 12 minutes, the volume fraction of mobile phase A changes from 68-70% to 18-20% at a constant rate, and the rest is mobile phase B;

[0028] From 12 to 17 minutes, the volume fraction of mobile phase A changes from 18-20% to 0% at a constant rate, and the rest is mobile phase B;

[0029] From 17 to 20 minutes, the volume fraction of mobile phase A changes from 0% to 98-100% at a constant rate, and the rest is mobile phase B;

[0030] From 20 to 24 minutes, the volume fraction of mobile phase A was from 98 to 100%, and the rest was mobile phase B.

[0031] In a preferred embodiment, the elution procedure in the chromatographic conditions is:

[0032]

[0033] Preferably, the hydrophobic interaction chromatography column in the chromatographic conditions is a liquid chromatography column for protein separation.

[0034] Preferably, the filler of the hydrophobic interaction chromatography column is functionalized silica gel particles.

[0035] Preferably, the hydrophobic interaction chromatography column is MAbPac HIC-10 produced by Thermo Fisher Scientific, with a pore size of 5 μm.

[0036] Preferably, the flow rate in the chromatographic conditions is 0.3-0.8 mL / min, for example, 0.3 mL / min, 0.5 mL / min or 0.8 mL / min.

[0037] Preferably, the column temperature in the chromatographic conditions is 25-30°C, for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0038] Preferably, the injection amount of the ADC drug in the chromatographic conditions is 10-40 μg, for example, 10 μg, 20 μg, 30 μg or 40 μg.

[0039] In the present invention, the ADC sample is directly injected, and the injection amount can be controlled by adjusting the injection volume according to the sample concentration. The injection amount of the ADC drug in the chromatographic conditions is 10-40 μg. In one embodiment of the present invention, the injection volume of the chromatograph is 1-100 μL; the specific injection volume is determined according to the chromatograph used.

[0040] Preferably, the method for calculating the average drug-antibody coupling ratio comprises:

[0041]

[0042] Preferably, the method for calculating the free small molecule content includes:

[0043]

[0044] Preferably, the liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content comprises:

[0045] The ADC drug sample was directly subjected to hydrophobic interaction chromatography detection, and the mobile phase included: mobile phase A: a mixed aqueous solution of 1.4-1.6M ammonium sulfate and 47-53mM potassium dihydrogen phosphate, pH 7.0±0.3; mobile phase B: a 47-53mM potassium dihydrogen phosphate aqueous solution containing 10-30% isopropanol; the hydrophobic interaction chromatography column was MAbPac HIC-10 from Thermo Fisher Scientific, with a pore size of 5μm; the flow rate was 0.3-0.8mL / min, the column temperature was 25-30°C, and the injection volume was 10-40μg; the elution program in the chromatographic conditions was:

[0046] In the first 0-1 minute, the volume fraction of mobile phase A was 98-100%, and the rest was mobile phase B;

[0047] In the first 3 minutes, the volume fraction of mobile phase A changes from 98-100% to 68-70% at a constant rate, and the rest is mobile phase B;

[0048] From 3 to 12 minutes, the volume fraction of mobile phase A changes from 68-70% to 18-20% at a constant rate, and the rest is mobile phase B;

[0049] From 12 to 17 minutes, the volume fraction of mobile phase A changes from 18-20% to 0% at a constant rate, and the rest is mobile phase B;

[0050] At 17-20 minutes, the volume fraction of mobile phase A changes from 0% to 98-100% at a constant rate, and the rest is mobile phase B;

[0051] From 20 to 24 minutes, the volume fraction of mobile phase A was 98-100%, and the rest was mobile phase B;

[0052] The detector collects wavelengths ranging from 270nm to 290nm and from 340nm to 390nm. The drug-antibody coupling ratio is calculated based on the data at a wavelength of 270nm to 290nm, and the peaks are classified as uncoupled antibody peaks, D2 peaks, D4 peaks, D6 peaks or D8 peaks. The proportion of each peak area reflects the distribution of the proportions of different coupling ratios, and the average drug-antibody coupling ratio is obtained by weighted calculation of the peak areas. The free small molecule content is calculated based on the data at a wavelength of 340nm to 390nm, and the peaks are classified as coupled antibody peaks and free small molecule peaks. The ratio of the peak area of ​​free small molecules to the total peak area is calculated to obtain the free small molecule content.

[0053] The technical solution of the present invention is based on a hydrophobic interaction chromatography that can simultaneously detect the drug-antibody coupling ratio, drug loading distribution and free small molecule content of ADC samples. The method is to directly inject the ADC sample, and the injection volume can be controlled by adjusting the injection volume according to the sample concentration, and the data within two wavelengths are collected for analysis at the same time. The optional wavelength range is divided into wavelength range 1: 270nm-290nm and wavelength range 2: 340nm-390nm, respectively measuring the drug-antibody coupling ratio, drug loading distribution and free small molecule content of the ADC sample, and reporting the key quality attributes of the ADC sample in a trinity.

[0054] In a second aspect, the present invention provides the use of the liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content described in the first aspect in detecting antibody-drug couplings.

[0055] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The method of the present invention can simultaneously report the drug-antibody coupling ratio, drug loading distribution and free small molecule content of the ADC sample in one experiment, greatly shortening the detection time.

[0058] (2) Samples can be injected directly without complicated operations such as precipitation and centrifugation, thus avoiding the impact of complex pretreatment on the samples.

[0059] (3) The free small molecule content can be directly reported by peak area ratio, without the need to use a more complicated external standard method for detection, which significantly reduces data processing time and improves detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The UV absorption spectra of naked antibody and ADC.

[0061] Figure 2 The chromatograms of ADC sample 1, ADC sample 2 and blank solution in Example 1 (collection wavelength 280 nm).

[0062] Figure 3 The chromatograms of ADC sample 1, ADC sample 2 and blank solution in Example 1 (collection wavelength 370 nm).

[0063] Figure 4 The chromatograms of ADC sample 1, ADC sample 2 and blank solution in Example 2 (collection wavelength 275 nm).

[0064] Figure 5 The chromatograms of ADC sample 1, ADC sample 2 and blank solution in Example 2 (collection wavelength 360 nm).

[0065] Figure 6 The chromatograms of ADC sample 3, ADC sample 4, ADC sample 5 and blank solution in Example 3 (collection wavelength 290 nm).

[0066] Figure 7 The chromatograms of ADC sample 3, ADC sample 4, ADC sample 5 and blank solution in Example 3 (collection wavelength 380 nm).

[0067] Figure 8 The chromatograms of the control sample, small molecule standard sample, spiked sample and blank solution in Example 4 (collection wavelength 370 nm).

[0068] Fig. 9 This is the chromatogram of Experiment 1 and Experiment 2 in Example 5 (collection wavelength 280nm).

[0069] Fig.10 This is the chromatogram of Experiment 1 and Experiment 2 in Example 5 (collection wavelength 370 nm).

[0070] Fig.11 The chromatograms of elution program 1, elution program 2 and elution program 3 in Example 6 (collection wavelength 370 nm). DETAILED DESCRIPTION

[0071] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0072] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0073] Example 1

[0074] 1. Sample preparation

[0075] (1) Blank solution

[0076] Take 100 μL of ultrapure water into the injection liner tube, and place the liner tube in a 2 mL liquid phase vial for later use.

[0077] (2) ADC sample solution

[0078] Take 100 μL of ADC sample 1 and ADC sample 2 respectively, transfer to the sample injection liner tube, and place in a 2 mL liquid phase vial for standby use. ADC sample 1 and ADC sample 2 are different samples obtained by coupling Herceptin monoclonal antibody with small molecule GGFG-DXD. ADC sample 1 and ADC sample 2 are prepared parallel samples obtained from the same batch.

[0079] 2. Chromatographic conditions

[0080] The chromatographic column MAbPac HIC-10, 4.6*100 mm, 5 μm, DAD detector, detector collection wavelength 280 nm and 370 nm, column temperature 25 ° C, flow rate 0.6 mL / min, injection volume 100 μg. The gradient elution program is shown in Table 1.

[0081] Mobile phase A: a mixed aqueous solution of 1.5 M ammonium sulfate and 50 mM potassium hydrogen phosphate, pH 7.0 ± 0.1;

[0082] Mobile phase B: 50 mM potassium phosphate dibasic aqueous solution containing 15% isopropanol.

[0083] Table 1

[0084] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.0 100 0 1.0 100 0 3.0 70 30 12.0 20 80 17.0 0 100 20.0 100 0 24.0 100 0

[0085] 3. Result calculation

[0086] (1) Drug-antibody coupling ratio

[0087] like Figure 2 As shown, the data at a wavelength of 280 nm are processed, and the peaks are respectively attributed to uncoupled antibodies, D2 (coupled with 2 small molecules), D4 (coupled with 4 small molecules), D6 (coupled with 6 small molecules) and D8 (coupled with 8 small molecules). The proportion of each peak area can reflect the distribution of the proportions of different coupling ratios. After weighted calculation of the peak area, the average drug-antibody coupling ratio can be obtained. The drug loading distribution is shown in Table 2. The main component is the D4 molecule. The drug-antibody coupling ratios of ADC sample 1 and ADC sample 2 are 3.9 and 3.9, respectively.

[0088] (2) Free small molecules

[0089] like Figure 3 As shown, the drug loading distribution at 370nm is shown. Figure 1It can be seen that since the small molecule contributes to all the ultraviolet absorption at this wavelength, the small molecule exists in two forms in the ADC sample, one is coupled to ADCs with different coupling ratios, and the other exists in a free form. The ratio of the peak area of ​​the free small molecule to the total peak area is the molar percentage content of the free small molecule (calculated in mol / mol), and the data are shown in Table 2. The free small molecule contents of ADC sample 1 and ADC sample 2 are 0.80% and 4.91%, respectively.

[0090] Table 2

[0091]

[0092] Example 2

[0093] 1. Sample preparation

[0094] (1) Blank solution

[0095] Take 100 μL of ultrapure water into the injection liner tube, and place the liner tube in a 2 mL liquid phase vial for later use.

[0096] (2) ADC sample solution

[0097] Take 100 μL of ADC sample 1 and ADC sample 2 respectively, transfer to the sample injection liner tube, and place in a 2 mL liquid phase vial for standby use. ADC sample 1 and ADC sample 2 are different samples obtained by coupling Herceptin monoclonal antibody with small molecule GGFG-DXD.

[0098] 2. Chromatographic conditions

[0099] The chromatographic column MAbPac HIC-10, 4.6*100 mm, 5 μm, DAD detector, detector collection wavelength 275 nm and 360 nm, column temperature 25 ° C, flow rate 0.6 mL / min, injection volume 100 μg. The gradient elution program is shown in Table 1.

[0100] Mobile phase A: a mixed aqueous solution of 1.5 M ammonium sulfate and 50 mM potassium hydrogen phosphate, pH 7.0 ± 0.1;

[0101] Mobile phase B: 50 mM potassium phosphate dibasic aqueous solution containing 15% isopropanol.

[0102] 3. Result calculation

[0103] (1) Drug-antibody coupling ratio

[0104] like Figure 4As shown, the data at a wavelength of 275 nm are processed, and the peaks are respectively attributed to uncoupled antibodies, D2 (coupled with 2 small molecules), D4 (coupled with 4 small molecules), D6 (coupled with 6 small molecules) and D8 (coupled with 8 small molecules). The proportion of each peak area can reflect the distribution of the proportions of different coupling ratios. After weighted calculation of the peak area, the average drug-antibody coupling ratio can be obtained. The drug loading distribution is shown in Table 3. The drug-antibody coupling ratios of ADC sample 1 and ADC sample 2 are 3.9 and 3.9, respectively. It is consistent with the coupling ratio results obtained in Example 1.

[0105] (2) Free small molecule content

[0106] like Figure 5 As shown, the drug loading distribution at 360nm is shown. Figure 1 It can be seen that since the small molecule contributes to all the ultraviolet absorption at this wavelength, the small molecule exists in two forms in the ADC sample, one is coupled to ADCs with different coupling ratios, and the other exists in a free form. The ratio of the peak area of ​​the free small molecule to the total peak area is the molar percentage content of the free small molecule (calculated in mol / mol), and the data are shown in Table 3. The free small molecule contents of ADC sample 1 and ADC sample 2 are 0.77% and 4.92%, respectively. It is comparable to the free small molecule content obtained in Example 1.

[0107] Table 3

[0108]

[0109] Example 3

[0110] 1. Sample preparation

[0111] (1) Blank solution

[0112] Take 100 μL of ultrapure water into the injection liner tube, and place the liner tube in a 2 mL liquid phase vial for later use.

[0113] (2) ADC sample solution

[0114] Take 100 μL of ADC sample 3, ADC sample 4 and ADC sample 5 respectively, transfer to the sample injection liner tube, and place in a 2 mL liquid phase vial for standby use. ADC sample 3, ADC sample 4 and ADC sample 5 are different samples obtained by coupling Herceptin monoclonal antibody with small molecule GGFG-DXD. ADC sample 3, ADC sample 4 and ADC sample 5 are samples obtained from three batches (three batches by changing reaction parameters).

[0115] 2. Chromatographic conditions

[0116] The chromatographic column MAbPac HIC-10, 4.6*100 mm, 5 μm, DAD detector, collection wavelengths of 290 nm and 380 nm, column temperature of 25°C, flow rate of 0.6 mL / min, injection volume of 100 μg were used. The gradient elution program is shown in Table 1.

[0117] Mobile phase A: a mixed aqueous solution of 1.5 M ammonium sulfate and 50 mM potassium hydrogen phosphate, pH 7.0 ± 0.1;

[0118] Mobile phase B: 50 mM potassium phosphate dibasic aqueous solution containing 15% isopropanol.

[0119] 3. Result calculation

[0120] (1) Drug-antibody coupling ratio

[0121] like Figure 6 As shown in Table 4, by processing the data at 290 nm, the peaks are respectively assigned to unconjugated antibodies, D2 (coupled with 2 small molecules), D4 (coupled with 4 small molecules), D6 (coupled with 6 small molecules) and D8 (coupled with 8 small molecules), and the proportion of each peak area can reflect the proportion of different coupling ratios. After weighted calculation of the peak area, the average drug-antibody coupling ratio can be obtained, and the data are shown in Table 4. The drug-antibody coupling ratios of ADC sample 3, ADC sample 4 and ADC sample 5 are 4.0, 3.5 and 2.7, respectively, indicating that this method can identify ADC samples with different coupling ratios.

[0122] (2) Free small molecule content

[0123] like Figure 7 As shown, the drug loading distribution at 380nm is shown. Figure 1 It can be seen that since small molecules contribute to all ultraviolet absorption at this wavelength, and small molecules exist in two forms in the ADC sample, one is coupled to ADCs with different coupling ratios, and the other exists in a free form. The ratio of the peak area of ​​the free small molecule to the total peak area is the molar percentage of the free small molecule (calculated in mol / mol). The data are shown in Table 4. The free small molecule percentages of ADC sample 3, ADC sample 4 and ADC sample 5 are 0.52%, 0.22% and 0.20%, respectively.

[0124] Table 4

[0125]

[0126] Example 4

[0127] 1. Sample preparation

[0128] (1) Blank solution

[0129] Take 50 μL of ultrapure water into the injection liner tube, and place the liner tube in a 2 mL liquid phase vial for later use.

[0130] (2) ADC control samples

[0131] Take 45 μL of ADC sample 6, add 5 μL of ultrapure water, mix thoroughly and transfer to the injection liner tube, and place in a 2 mL liquid phase vial for later use. ADC sample 6 is obtained by coupling Herceptin monoclonal antibody with small molecule GGFG-DXD.

[0132] (3) ADC spiked samples

[0133] Take 45 μL of ADC sample 6, add 5 μL of small molecule solution with a concentration of 20 μg / mL, mix thoroughly and transfer to the injection liner tube, and place in a 2 mL liquid phase vial for use.

[0134] (4) Small molecule solution

[0135] Take 5 μL of a small molecule solution with a concentration of 20 μg / mL, add 45 μL of ultrapure water to dilute to the target concentration of 2 μg / mL. After thorough mixing, transfer to the injection liner tube and place in a 2 mL liquid phase vial for later use.

[0136] 2. Chromatographic conditions

[0137] The chromatographic column MAbPac HIC-10, 4.6*100 mm, 5 μm, DAD detector, detector collection wavelength 275 nm and 360 nm, column temperature 25°C, flow rate 0.6 mL / min, injection volume 10 μL. The gradient elution program is shown in Table 1.

[0138] Mobile phase A: a mixed aqueous solution of 1.5 M ammonium sulfate and 50 mM potassium hydrogen phosphate, pH 7.0 ± 0.1;

[0139] Mobile phase B: 50 mM potassium phosphate dibasic aqueous solution containing 15% isopropanol.

[0140] 3. Free small molecule content

[0141] like Figure 8 As shown, the spectra of ADC control sample, ADC spiked sample and small molecule solution at 370nm are shown. The peak area results of ADC control sample, small molecule sample and ADC spiked sample are shown in Table 5. The recovery rate is calculated by peak area, and it can be obtained that the recovery rate is 97.1%, indicating that this method can well detect the content of free small molecules in ADC samples.

[0142] Table 5

[0143]

[0144] Example 5

[0145] This example investigates the mobile phase in the liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content.

[0146] Experiment 1:

[0147] Mobile phase A: a mixed aqueous solution of 1.5 M ammonium sulfate and 50 mM potassium hydrogen phosphate, pH 7.0;

[0148] Mobile phase B: 50 mM potassium hydrogen phosphate in water containing 20% ​​isopropanol.

[0149] Experiment 2:

[0150] Mobile phase A: a mixed aqueous solution of 1.5 M ammonium sulfate and 50 mM potassium hydrogen phosphate, pH 7.0;

[0151] Mobile phase B: 50 mM potassium hydrogen phosphate aqueous solution containing 20% ​​acetonitrile.

[0152] The results of the investigation are shown in Table 6 and Fig. 9 and Fig.10 shown.

[0153] Table 6

[0154]

[0155] From the investigation results, it can be seen that acetonitrile or isopropanol can be added as the organic solvent in mobile phase B to simultaneously detect the ADC drug-antibody coupling ratio and the content of free small molecules. Among them, when isopropanol is used as an organic additive, the free small molecule content can be more significantly separated and detected. Fig.10 From the spectra under different conditions, it can be seen that the free drug peak in Experiment 1 is more obvious, and isopropanol is the preferred organic additive.

[0156] Example 6

[0157] This example investigates the elution procedure in the liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content. The samples are samples of the same type.

[0158] Elution program 1 is shown in Table 7.

[0159] Table 7

[0160] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 1 100 0 3 70 30 15 30 70 18 0 100 20 100 0 21 100 0

[0161] Elution program 2 is shown in Table 8.

[0162] Table 8

[0163] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 1 100 0 3 70 30 12 20 80 15 0 100 16 100 0 18 100 0

[0164] Elution program 3 is shown in Table 9.

[0165] Table 9

[0166] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 1 100 0 3 70 30 12 20 80 17 0 100 20 100 0 24 100 0

[0167] from Fig.11 It can be seen that the separation degree in elution program 1 is poor, and it is impossible to separate the relevant components and calculate the antibody coupling ratio. Elution programs 2 and elution programs 3 show good separation degrees. Therefore, the preferred range of elution programs is elution program 3, the core of which is to reduce the gradient of mobile phase A from 70% to 20% within 3-12 minutes.

[0168] In summary, the present invention provides a method for simultaneously detecting the drug-antibody coupling ratio and free small molecule content of ADC samples based on hydrophobic interaction chromatography. The method can be directly injected during detection without protein precipitation and external standard, and the drug-antibody coupling ratio and drug loading distribution of the sample, and the content of free small molecules are detected simultaneously. The method of the present invention greatly shortens the detection time, simplifies the detection steps, and avoids the influence of complex pretreatment on the sample. In particular, the free small molecule content can be directly reported by the peak area ratio, without the need to use a more complex external standard method for detection, which significantly reduces the data processing time and improves the detection efficiency.

[0169] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A liquid chromatography method for simultaneously detecting ADC drug-antibody coupling ratio and free small molecule content, characterized in that: The method comprises: using hydrophobic interaction chromatography to detect and analyze ADC drug samples, wherein the detector collection wavelength range in the chromatographic conditions includes 270nm-290nm and 340nm-390nm; The drug-antibody coupling ratio is calculated based on the data of 270nm-290nm wavelength, and the peaks are classified as uncoupled antibody peaks, D2 peaks, D4 peaks, D6 peaks or D8 peaks; the proportion of each peak area reflects the distribution of different coupling ratios, and the average drug-antibody coupling ratio is obtained by weighted calculation of the peak area; The free small molecule content was calculated based on the data at a wavelength of 340nm-390nm, the peaks were attributed to the coupled antibody peaks and the free small molecule peaks, and the ratio of the peak area of ​​the free small molecule to the total peak area was calculated to obtain the free small molecule content.

2. The liquid chromatography method for simultaneously detecting ADC drug-antibody coupling ratio and free small molecule content according to claim 1, characterized in that: The small molecule type in the ADC is a Dxd type small molecule.

3. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to claim 1 or 2, characterized in that: The mobile phase in the chromatographic conditions comprises: Mobile phase A: a mixed aqueous solution of 1.4-1.6 M ammonium sulfate and 47-53 mM potassium dihydrogen phosphate, pH 7.0 ± 0.3; Mobile phase B: 47-53 mM potassium dihydrogen phosphate aqueous solution containing 10-30% isopropanol; Preferably, the elution procedure in the chromatographic conditions comprises: In the first 0-1 minute, the volume fraction of mobile phase A was 98-100%, and the rest was mobile phase B; In the first 3 minutes, the volume fraction of mobile phase A changes from 98-100% to 68-70% at a constant rate, and the rest is mobile phase B; From 3 to 12 minutes, the volume fraction of mobile phase A changes from 68-70% to 18-20% at a constant rate, and the rest is mobile phase B; From 12 to 17 minutes, the volume fraction of mobile phase A changes from 18-20% to 0% at a constant rate, and the rest is mobile phase B; From 17 to 20 minutes, the volume fraction of mobile phase A changes from 0% to 98-100% at a constant rate, and the rest is mobile phase B; From 20 to 24 minutes, the volume fraction of mobile phase A was from 98 to 100%, and the rest was mobile phase B.

4. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 3, characterized in that: The hydrophobic interaction chromatography column in the chromatographic conditions is a liquid chromatography column used for protein separation; Preferably, the filler of the hydrophobic interaction chromatography column is functionalized silica gel particles; Preferably, the hydrophobic interaction chromatography column is MAbPac HIC-10 produced by Thermo Fisher Scientific, with a pore size of 5 μm.

5. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 4, characterized in that: The flow rate in the chromatographic conditions is 0.3-0.8 mL / min.

6. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 5, characterized in that: The column temperature in the chromatographic conditions is 25-30°C.

7. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 6, characterized in that: The injection amount of the ADC drug in the chromatographic conditions is 10-40 μg.

8. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 7, characterized in that: The calculation method of the average drug-antibody coupling ratio DAR includes: Preferably, the method for calculating the free small molecule content includes:

9. The liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 8, characterized in that: The method comprises: The ADC drug sample was subjected to hydrophobic interaction chromatography, and the mobile phase included: mobile phase A: a mixed aqueous solution of 1.4-1.6M ammonium sulfate and 47-53mM potassium dihydrogen phosphate, pH 7.0±0.3; mobile phase B: a 47-53mM potassium dihydrogen phosphate aqueous solution containing 10-30% isopropanol or acetonitrile; the hydrophobic interaction chromatography column was MAbPac HIC-10 produced by Thermo Fisher, and the filler was functionalized silica gel particles with a pore size of 5μm; the flow rate was 0.3-0.8mL / min, the column temperature was 25-30°C, and the injection volume was 10-40μg; the elution program in the chromatographic conditions was: In the first 0-1 minute, the volume fraction of mobile phase A was 98-100%, and the rest was mobile phase B; In the first 3 minutes, the volume fraction of mobile phase A changes from 98-100% to 68-70% at a constant rate, and the rest is mobile phase B; From 3 to 12 minutes, the volume fraction of mobile phase A changes from 68-70% to 18-20% at a constant rate, and the rest is mobile phase B; From 12 to 17 minutes, the volume fraction of mobile phase A changes from 18-20% to 0% at a constant rate, and the rest is mobile phase B; From 17 to 20 minutes, the volume fraction of mobile phase A changes from 0% to 98-100% at a constant rate, and the rest is mobile phase B; From 20 to 24 minutes, the volume fraction of mobile phase A was 98-100%, and the rest was mobile phase B; The detector collects wavelengths ranging from 270nm to 290nm and from 340nm to 390nm. The drug-antibody coupling ratio is calculated based on the data at a wavelength of 270nm to 290nm, and the peaks are classified as uncoupled antibody peaks, D2 peaks, D4 peaks, D6 peaks or D8 peaks. The proportion of each peak area reflects the distribution of the proportions of different coupling ratios, and the average drug-antibody coupling ratio is obtained by weighted calculation of the peak areas. The free small molecule content is calculated based on the data at a wavelength of 340nm to 390nm, and the peaks are classified as coupled antibody peaks and free small molecule peaks. The ratio of the peak area of ​​free small molecules to the total peak area is calculated to obtain the free small molecule content.

10. Use of the liquid chromatography method for simultaneously detecting the ADC drug-antibody coupling ratio and the free small molecule content according to any one of claims 1 to 9 in detecting antibody-drug couplings.

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