Method for detecting antibody protein content in antibody conjugate drugs

Abstract

The application provides a method for detecting the content of antibody protein in antibody conjugate drugs. The antibody conjugate drug comprises an antibody protein and a toxin linker. The method comprises the following steps: detecting the chromatographic peaks of the toxin linker at wavelength 1 and wavelength 2 by using chromatography, calculating the area ratio of the two chromatographic peaks of the toxin linker at wavelength 1 and wavelength 2, and obtaining the content of the antibody protein according to the ratio. The method can solve the problem of low accuracy of the result of detecting the content of antibody protein in antibody conjugate drugs in the prior art, and is suitable for the field of detecting antibody conjugate drugs.

Description

Technical Field

[0001] This invention relates to the field of antibody-drug conjugate detection, and more specifically, to a method for detecting the content of antibody proteins in antibody-drug conjugates. Background Technology

[0002] Antibody protein content is one of the most frequently measured quality parameters when analyzing key quality attributes of antibody-drug conjugates (ADCs). Because the antibody protein content of ADC samples significantly impacts the quality, potency, and safety of ADC drugs, it is necessary to determine protein content during ADC manufacturing, release testing of ADC bulk solutions / formulations, and stability studies. In biopharmaceutical preparation experiments, common methods for detecting ADC protein content include ultraviolet (UV) spectrophotometry and the BCA (Bicinchoninic Acid) method.

[0003] Ultraviolet (UV) spectrophotometry plays a crucial role in the protein content detection of antibody-drug conjugates (ADCs) due to its simplicity, speed, low variability, and high accuracy. However, accurate determination of ADC protein content using UV methods relies on mastering the precise extinction coefficient of the antibody protein and toxin linker at a specific wavelength. While the extinction coefficient of the antibody protein can be obtained through theoretical calculations or experimental measurements of pure antibody protein, the determination of the extinction coefficient of the toxin linker coupled to the antibody generally requires a high-purity standard of known concentration of free toxin linker (i.e., uncoupled toxin linker) obtained via UV-Vis spectroscopy. This method is often limited by the following factors, making it impossible to obtain the necessary toxin linker extinction coefficient, thus preventing the use of UV methods to measure ADC protein content:

[0004] First, the purity of toxin linkers is low, containing impurities that produce UV absorption, making it difficult to obtain pure standards. The cuvette UV method lacks the ability to identify impurities, and the measured absorbance is affected by the absorption of non-specific impurities, leading to distortion in the determination of the toxin linker extinction coefficient. This problem is particularly prominent in the early stages of toxin linker development, when the synthetic routes and purification techniques are still unstable. This directly affects subsequent process development and product quality assessment, such as yield estimation, purification strategy formulation, and coupling condition optimization. It also introduces significant uncertainty and bias into other analytical tests that rely on protein content results, such as activity assays.

[0005] Secondly, some toxin linkers contain specific functional groups, such as phenyl isothiocyanate for lysine coupling and methyl sulfone heterocyclic aromatic hydrocarbons for cysteine ​​coupling. After these groups chemically couple with the corresponding sites on the antibody, their UV absorption spectra change significantly, differing from the uncoupled state. Therefore, directly using the extinction coefficient of the uncoupled toxin linker cannot accurately reflect the UV absorption characteristics of the coupled toxin linker, leading to significant errors in protein content calculations.

[0006] Given these two major obstacles, in practice, the accuracy of UV method measurements cannot be guaranteed in many cases, necessitating a switch to other assays such as BCA (Bicinchoninic Acid). However, these alternative methods involve complex sample pretreatment processes, long detection cycles, and significant internal variability, resulting in poor accuracy. This greatly increases the difficulty of control in the production process and the risk of out-of-specification (OOS) defects in finished products, sometimes even leading to the scrapping of the entire batch of ADC products.

[0007] In summary, the current UV method for determining ADC protein content faces the dual challenges of insufficient purity of toxic linkers and spectral changes after coupling. This not only restricts the efficiency of ADC production but also affects the quality and safety of the final product. Summary of the Invention

[0008] The main objective of this invention is to provide a method for detecting the antibody protein content in antibody-drug conjugates, thereby solving the problem of low accuracy in the detection of antibody protein content in antibody-drug conjugates in the prior art.

[0009] To achieve the above objectives, according to a first aspect of the present invention, a method for detecting the content of antibody protein in an antibody-drug conjugate is provided, the antibody-drug conjugate comprising antibody protein and toxin linker;

[0010] The detection method includes: using chromatography to detect the chromatographic peaks of the toxin linker at wavelengths 1 and 2, and calculating the ratio of the areas of the two chromatographic peaks of the toxin linker at wavelengths 1 and 2; calculating the content of the antibody protein based on the ratio; wavelength 1 is the characteristic absorption wavelength of the antibody-drug conjugate, antibody protein, or toxin linker between 200 and 300 nm; correspondingly, wavelength 2 is the characteristic absorption wavelength of the antibody-drug conjugate, antibody protein, or toxin linker between 250 and 400 nm; the values ​​of wavelength 1 and wavelength 2 are different.

[0011] Furthermore, the antibody protein content is calculated using the following formula:

[0012] ;

[0013] The subscripts w1 and w2 represent two wavelengths respectively;

[0014] Where k represents the ratio of the areas of the two chromatographic peaks of the toxin linker at wavelengths 1 and 2;

[0015] A w1*ADC and A w2*ADC These are the absorbance values ​​of the antibody-drug conjugate at wavelengths 1 and 2, respectively.

[0016] and This indicates the molar extinction coefficient of the antibody protein at wavelengths 1 and 2;

[0017] C mAb This refers to the content of antibody protein in the antibody-drug conjugate, i.e., molar concentration.

[0018] L is the optical path length of the cuvette;

[0019] DF is the dilution factor.

[0020] Further, the detection method includes: S1) dissolving the toxin linker in a solvent to obtain a solution of the toxin linker; S2) injecting the solution of the toxin linker into a chromatogram to separate the toxin linker from impurities in the solution, obtaining two chromatographic peaks of the toxin linker at wavelength 1 and wavelength 2; calculating the ratio of the peak areas of the two chromatographic peaks; S3) substituting the ratio, the absorbance of the antibody-drug conjugate at wavelength 1 and wavelength 2, and the molar extinction coefficient of the antibody protein at wavelength 1 and wavelength 2 into the above formula to calculate the content of antibody protein in the antibody-drug conjugate.

[0021] Furthermore, the detection method also includes: a1) dissolving the toxin linker in a solvent to obtain a solution of the toxin linker; a2) mixing the solution of the toxin linker with the coupling molecule for coupling to obtain a solution of the first coupling compound; a3) injecting the solution of the first coupling compound into a chromatogram to separate the first coupling compound from impurities in the solution of the first coupling compound, and obtaining two chromatographic peaks of the first coupling compound at wavelength 1 and wavelength 2; calculating the ratio of the peak areas of the two chromatographic peaks; a4) substituting the ratio, the absorbance of the antibody-drug conjugate at wavelength 1 and wavelength 2, and the molar extinction coefficient of the antibody protein at wavelength 1 and wavelength 2 into the above formula to calculate the content of antibody protein in the antibody-drug conjugate.

[0022] Furthermore, the coupling molecules include amino-containing compounds and / or thiol-containing compounds.

[0023] Furthermore, amino-containing compounds include any one or more of the following: i) compounds containing both amino and carboxyl groups; ii) amine compounds.

[0024] Furthermore, compounds containing amino and carboxyl groups include amino acids and / or aminocaproic acid; amine compounds include methylamine and / or ethylamine.

[0025] Furthermore, the thiol-containing compounds include one or more of acetylcysteine, cysteine, or glutathione.

[0026] Furthermore, in a2), the molar ratio of the toxin linker to the coupling molecule is 1:5~20.

[0027] Furthermore, the chromatography includes one or more of the following: reversed-phase chromatography, normal-phase chromatography, hydrophilic-interaction chromatography, hydrophobic chromatography, ion-exchange chromatography, or capillary electrophoresis.

[0028] Using the technical solution of this invention, high-purity toxin linkers are not required as standards. In the process of detecting the antibody protein content in antibody-drug conjugates (ADCs), the chromatographic peaks of the toxin linker at wavelengths 1 (200-300 nm) and 2 (250-400 nm) are detected by chromatography, and the ratio of the areas of the two peaks is calculated. This ratio is then substituted into the formula for calculating the antibody protein content to obtain the antibody protein content. The detection method of this application can rapidly and accurately detect the antibody protein content in ADCs, improving the accuracy of antibody protein content detection in ADCs. It has low sample requirements, a simple operation process, and lowers the technical application threshold, providing a data foundation and technical support for the early stages of ADC process development. Attached Figure Description

[0029] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0030] Figure 1 A schematic diagram of the reaction between the toxin linker and the antibody in Example 1 according to this application specification is shown.

[0031] Figure 2 A schematic diagram of the reaction between the toxin linker and the coupling molecule in Example 1 according to this application specification is shown.

[0032] Figure 3 The UV absorption spectra of the free toxin linker and the toxin linker-aminocaproic acid conjugate according to Example 1 of this application are shown; wherein, Figure 3 Image A is the UV absorption spectrum of the toxin linker-6-aminohexanoic acid conjugate (the first conjugate). Figure 3 B is the UV absorption spectrum of the toxin linker.

[0033] Figure 4The chromatograms of the toxin linker-aminocaproic acid conjugate at wavelengths 1 and 2 in Example 1 according to this application are shown; wherein, Figure 4 Chromatogram A is the chromatogram of the toxin linker aminocaproic acid conjugate (first conjugate) detected at a wavelength of 250 nm; Figure 4 Chromatogram B is the chromatogram of the toxin linker aminocaproic acid conjugate (first conjugate) detected at a wavelength of 280 nm.

[0034] Figure 5 A schematic diagram of the reaction between the toxin linker and the antibody in Example 2 according to this application specification is shown.

[0035] Figure 6 A schematic diagram of the reaction between the toxin linker and the coupling molecule in Example 2 according to this application specification is shown.

[0036] Figure 7 The UV absorption spectra of the free toxin linker and the toxin linker-acetylcysteine ​​conjugate according to Example 2 of this application are shown; wherein, Figure 7 Image A shows the UV absorption spectrum of the toxin linker-acetylcysteine ​​conjugate (the first conjugate). Figure 7 B is the UV absorption spectrum of the toxin linker.

[0037] Figure 8 The chromatograms of the toxin linker-acetylcysteine ​​conjugate at wavelengths 1 and 2 in Example 2 according to this application are shown; wherein, Figure 8 Chromatogram A is the chromatogram of the toxin linker-acetylcysteine ​​conjugate (first conjugate) detected at a wavelength of 260 nm. Figure 8 Chromatogram B is the chromatogram of the toxin linker-acetylcysteine ​​conjugate (first conjugate) detected at a wavelength of 280 nm. Detailed Implementation

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0039] As mentioned in the background art, it is difficult to obtain high-purity toxin linker standards and the extinction coefficient cannot be accurately reflected after the toxin linker is coupled, resulting in low accuracy of traditional UV methods for determining the antibody protein content of antibody-drug conjugates. Therefore, in this application, the inventors attempted to develop a new method for detecting the antibody protein content in antibody-drug conjugates, and thus proposed a series of protection schemes in this application.

[0040] In a first typical embodiment of this application, a method for detecting the content of antibody protein in an antibody-drug conjugate is provided. The antibody-drug conjugate includes an antibody protein and a toxin linker. The detection method includes: detecting the chromatographic peaks of the toxin linker at wavelengths 1 and 2 using chromatography, and calculating the ratio of the areas of the two chromatographic peaks of the toxin linker at wavelengths 1 and 2; calculating the content of the antibody protein based on the ratio; wavelength 1 is the characteristic absorption wavelength of the antibody-drug conjugate, antibody protein, or toxin linker between 200 and 300 nm; correspondingly, wavelength 2 is the characteristic absorption wavelength of the antibody-drug conjugate, antibody protein, or toxin linker between 250 and 400 nm; the values ​​of wavelength 1 and wavelength 2 are different.

[0041] Determining the antibody protein content in antibody-drug conjugates (ADCs) is a crucial step in the biopharmaceutical field. Accurate protein content data directly impacts the efficacy and safety of ADCs, i.e., the precise dosage in targeted therapy, and is also essential for drug development, manufacturing, and quality control. In ADC quality control, the UV method, with its ease of operation, speed, and high precision, has become the preferred analytical approach. However, due to the limitations of traditional UV methods, accurate and reliable antibody protein content data are difficult to obtain when the purity of the toxin linker is insufficient or when its UV absorption characteristics are significantly altered after conjugation with the antibody protein. This poses a significant challenge to product and process development in the early stages of ADC research and development.

[0042] This application presents a chromatographic detection method that requires fewer parameters to be measured and is simple in method. It can detect the ratio of the extinction coefficients of toxin linkers at two wavelengths without the need for high-purity toxin linker standards, thus avoiding more complex chromatographic and mass spectrometric methods. It can quickly and accurately detect the content of ADC antibody proteins, improve the overall R&D efficiency, production quality and safety of clinical applications of ADC drugs, and further promote technological progress and drug innovation in the ADC field.

[0043] This application creatively combines the separation advantages of liquid chromatography with the rapid response of UV detection, breaking through the bottleneck of transition from spectroscopy to chromatography. It not only solves the detection difficulties caused by changes in the purity of toxin linkers and UV absorption characteristics after coupling, but also expands the application scope and accuracy of UV method in the determination of ADC protein content, achieving a technological breakthrough and providing a more stable and efficient solution for the analysis and detection of ADC drugs, with high scientific and technological value.

[0044] The detection method of this application specifically includes: separating the chromatographic peak of the toxin linker from its impurities using chromatography; simultaneously collecting chromatographic signals at wavelength 1 and wavelength 2 using an ultraviolet detector (hereinafter referred to as UV detector) in liquid chromatography to obtain chromatograms of the pure toxin linker separated by chromatography at two wavelengths; obtaining the peak area of ​​the toxin linker in the chromatograms at two different wavelengths by peak integration; and calculating the ratio, which can represent the ratio of the extinction coefficients of the toxin linker obtained by the traditional UV dual-wavelength method. Substituting the ratio of the two peak areas obtained in this application into the formula for determining the ADC protein content by the UV dual-wavelength method, the calculated ADC protein content data is not much different from the theoretical value, the data accuracy is high, and the error is small.

[0045] This application uses a chromatographic detection method that avoids unnecessary errors caused by sample pretreatment and impurity interference in the traditional UV dual-wavelength method based on spectral determination, as well as its dependence on high-purity toxin linker samples. It simplifies the data conversion steps and provides accurate and reliable detection results. Compared with the traditional UV dual-wavelength method, the detection method of this application has high detection efficiency, low sample requirements, and high detection accuracy, making it more suitable for application and promotion in the ADC field.

[0046] It should be noted that, for the sake of simplicity in describing the relevant parameters in this application, some parameters are represented by abbreviations or letters, such as... , , , The use of abbreviations such as "k" is not intended to limit the actual scope. Through the above explanation, this application aims to emphasize that the use of abbreviations and letter symbols should not be considered a limitation on the scope of the technical solution, but rather a way of expressing the technology in a more concise and easily understood manner.

[0047] In a preferred embodiment, the antibody protein content is calculated using the following formula:

[0048] ;

[0049] The subscripts w1 and w2 represent two wavelengths; where k represents the ratio of the areas of the two chromatographic peaks of the toxin linker at wavelengths 1 and 2; A w1*ADC A w2*ADC These are the absorbance values ​​of the antibody-drug conjugate at wavelengths 1 and 2, respectively. and , represents the molar extinction coefficient of the antibody protein at wavelengths 1 and 2; C mAb denoted as L, representing the antibody protein content in the antibody-drug conjugate; L represents the optical path length of the cuvette; and DF represents the dilution factor.

[0050] An ADC molecule consists of two parts: an antibody protein and a toxin linker. These two components are covalently linked (i.e., coupled) to form a complete ADC molecule. Generally, the antibody and toxin linker exhibit different UV absorption spectral characteristics. Therefore, at a specific wavelength (w), the absorbance of an ADC molecule is the sum of the absorbances of the antibody and the toxin linker. There is a linear relationship between absorbance and the concentration (content) of the absorbant component, i.e., it follows the Lambert-Beer Law.

[0051] A = εbc (where A is absorbance, ε is extinction coefficient, b is optical path length, and c is molar concentration). The derivation of the formula for calculating antibody protein content using the Lambert-Beer law is shown below:

[0052] ;

[0053] ;

[0054] ;

[0055] ;

[0056] In the above formula, , The molar extinction coefficients of the toxin linker in the ADC at wavelengths 1 and 2;

[0057] C PL : Molar concentration of toxin linkers in ADC.

[0058] In a preferred embodiment, the detection method includes: S1) dissolving the toxin linker in a solvent to obtain a solution of the toxin linker; S2) injecting the solution of the toxin linker into a chromatogram to separate the toxin linker from impurities in the solution, obtaining two chromatographic peaks of the toxin linker at wavelength 1 and wavelength 2; calculating the ratio of the peak areas of the two chromatographic peaks; S3) substituting the ratio, the absorbance of the antibody-drug conjugate at wavelength 1 and wavelength 2, and the molar extinction coefficient of the antibody protein at wavelength 1 and wavelength 2 into the above formula to calculate the content of antibody protein in the antibody-drug conjugate.

[0059] In traditional UV detection methods, accurately determining the antibody protein content in an ADC requires independently obtaining the molar concentrations of the antibody and toxin linker, a complex process easily affected by sample purity. The detection method proposed in this application separates the toxin linker from impurities using liquid chromatography or capillary electrophoresis, reducing interference from impurities coexisting with the toxin linker on the determination of its extinction coefficient, and avoiding the influence of purity on the accuracy of the detection results.

[0060] This application directly obtains the ratio of the chromatographic peak areas of the toxin linker molecule at two specific wavelengths after chromatographic detection by setting the UV detector to operate at two specific wavelengths. Since the peak area is proportional to the extinction coefficient and is based on the same injection, the peak area ratio is the ratio of the extinction coefficients of the toxin linker molecule at the two wavelengths. Once the ratio is obtained, the protein content in the ADC sample can be calculated. This detection method simplifies the data conversion steps, avoids unnecessary errors caused by sample pretreatment and impurity interference in traditional methods, reduces the need for high-purity toxin linker samples, reduces detection costs, and improves the accuracy and reliability of the calculation process and results.

[0061] If the absorption characteristics of the toxin linker do not change before and after coupling with the antibody protein at the two wavelengths, the specific method for obtaining the ratio k of the absorbance coefficients at wavelength 1 and wavelength 2 includes:

[0062] 1) Prepare the toxin linker sample solution (i.e., the toxin linker solution mentioned above). The concentration is preferably around 0.1~1 mg / mL. The exact concentration is not required. It is sufficient to obtain a strong toxin linker chromatographic peak (the signal-to-noise ratio of the chromatographic peak is greater than 200). Those skilled in the art can adjust it flexibly according to the actual situation.

[0063] 2) Configure the chromatogram and set up dual-wavelength detection using a UV detector;

[0064] The preferred chromatographic method is liquid chromatography (LC). The preferred chromatographic conditions are as follows: a C18 reversed-phase column is connected to the LC column; water containing 0.1% TFA and acetonitrile containing 0.1% TFA are used as the mobile phase; a gradient elution program is set (acetonitrile concentration increases from 5% to 40% within 10 minutes); 250 nm and 280 nm are set as the detection wavelengths for LC; and a diode array detector (DAD) is used for full wavelength scanning in the range of 200 nm to 400 nm. Those skilled in the art can flexibly adjust the chromatographic conditions according to actual conditions.

[0065] 3) Inject and analyze the toxin linker sample solution, in which the toxin linker is separated from other impurities, and chromatograms of the toxin linker at two wavelengths are obtained simultaneously;

[0066] 4) Integrate the chromatographic peaks of the toxin linker to obtain the peak areas at the two wavelengths, and calculate the ratio (i.e., the k value mentioned above).

[0067] 5) Substitute the k value into the ADC protein content calculation formula to calculate the antibody protein content.

[0068] In a preferred embodiment, the detection method further includes: a1) dissolving the toxin linker in a solvent to obtain a solution of the toxin linker; a2) mixing the solution of the toxin linker with the coupling molecule for coupling to obtain a solution of the first coupling compound; a3) injecting the solution of the first coupling compound into a chromatogram to separate the first coupling compound from impurities in the solution of the first coupling compound, and obtaining two chromatographic peaks of the first coupling compound at wavelength 1 and wavelength 2; calculating the ratio of the peak areas of the two chromatographic peaks; a4) substituting the ratio, the absorbance of the antibody-drug conjugate at wavelength 1 and wavelength 2, and the molar extinction coefficient of the antibody protein at wavelength 1 and wavelength 2 into the above formula to calculate the content of antibody protein in the antibody-drug conjugate.

[0069] As is known in the background art, the molecular structure of certain toxin linkers changes after conjugation with antibodies, often leading to significant changes in their UV absorption characteristics at a selected wavelength. In this case, directly using the k-value measured by the toxin linker before conjugation to calculate the protein content of the ADC will not accurately reflect the UV absorption characteristics of the toxin linker after conjugation, resulting in a significant error in the protein content calculation. The method for preparing the toxin linker-small molecule conjugate described above in this application, i.e., the method for forming the first conjugate described above, can eliminate this effect and obtain an accurate k-value. The specific steps of the determination method in this case include:

[0070] 1) Prepare the toxin linker solution, preferably at a concentration of about 0.1~1 mg / mL. The exact concentration is not required here, as long as a strong toxin linker chromatographic peak is obtained (chromatographic peak signal-to-noise ratio greater than 200). Those skilled in the art can adjust it flexibly according to the actual situation.

[0071] 2) Preparation of toxin linker-small molecule conjugate (first conjugate): Using a buffer system consistent with the ADC coupling reaction, toxin linker and excess of simulated small chemical molecules (coupling molecules) that can react with toxin linker are added, and the two are mixed to carry out the coupling reaction to obtain a solution of the first conjugate.

[0072] 3) Configure the chromatography; the UV detector needs to be set to dual-wavelength detection.

[0073] The preferred chromatographic method is liquid chromatography (LC). Chromatographic conditions include, but are not limited to, connecting a C18 reversed-phase column to the LC system, using water containing 0.1% TFA and acetonitrile containing 0.1% TFA as the mobile phase, setting a gradient elution program (acetonitrile concentration increasing from 20% to 70% within 10 minutes), setting 260 nm and 280 nm as the detection wavelengths for LC, and simultaneously setting a diode array detector (DAD) to perform a full wavelength scan in the range of 200 nm to 400 nm. Alternatively, connecting a C18 reversed-phase column to the LC system, using water containing 0.1% TFA and acetonitrile containing 0.1% TFA as the mobile phase, setting a gradient elution program (acetonitrile concentration increasing from 5% to 40% within 10 minutes), setting 250 nm and 280 nm as the detection wavelengths for LC, and simultaneously setting a diode array detector (DAD) to perform a full wavelength scan in the range of 200 nm to 400 nm. Those skilled in the art can flexibly adjust the chromatographic conditions according to actual conditions.

[0074] 4) The toxin linker-small molecule conjugate solution obtained in step 2 was injected for analysis. The toxin linker-small molecule conjugate was separated from other impurities, and chromatograms of the toxin linker-small molecule conjugate at two wavelengths were obtained.

[0075] Since liquid chromatography can separate toxin linker-small molecule conjugates from other impurities (including toxin linkers that have not undergone coupling reaction and excess small molecules), the reaction solution obtained in step 2 (i.e. the solution of the first conjugate) does not require any pre-purification treatment and can be directly subjected to chromatographic injection analysis.

[0076] 5) Integrate the chromatographic peaks of the toxin linker-small molecule conjugate to obtain the peak areas at two wavelengths and calculate the ratio (k value).

[0077] 6) Substitute the k value into the ADC protein content calculation formula to calculate the protein content.

[0078] As mentioned above, the reaction solution of the toxin linker-small molecule conjugate (first conjugate) is a complex mixture containing unreacted toxin linkers and small molecule conjugates, as well as complex byproducts. Therefore, ultraviolet spectroscopy cannot determine the extinction coefficient of the first conjugate at two wavelengths. However, the liquid chromatography separation method of this application allows for the determination of the chromatographic peak areas of the first conjugate at two wavelengths. The ratio of the two peak areas can be considered as the k value in the above formula and substituted into the formula to accurately calculate the antibody protein content in the ADC.

[0079] In a preferred embodiment, the coupling molecule includes an amino (-NH2) compound and / or a mercapto (-SH) compound.

[0080] In a preferred embodiment, the amino-containing compound includes any one or more of the following: a) compounds containing both amino and carboxyl groups; b) amine compounds.

[0081] In a preferred embodiment, the compounds containing amino and carboxyl groups include amino acids and / or aminocaproic acid; the amine compounds include methylamine and / or ethylamine.

[0082] In a preferred embodiment, the thiol-containing compound includes one or more of acetylcysteine, cysteine, or glutathione.

[0083] This application overcomes the problem that changes in the UV absorption characteristics of the toxin linker in antibody-drug conjugates (ADCs) before and after conjugation affect the accuracy of determining the antibody protein content in ADCs. To accurately determine the ratio of the extinction coefficients of the toxin linker conjugated to the antibody at two specific wavelengths, this application introduces a technique that simulates the conjugation reaction using small molecules. Specifically, based on the conjugation type between the antibody and the toxin linker in the ADC, small molecules capable of mimicking functional groups on the antibody are selected. For example, for lysine-conjugated toxin linkers, the conjugating molecule reacting with it can be 6-aminocaproic acid; for cysteine-conjugated toxin linkers, the conjugating molecule reacting with it can be acetylcysteine. These conjugating molecules have no absorption at the selected UV wavelength, ensuring that they do not interfere with the accurate determination of the UV absorption characteristics of the toxin linker.

[0084] By performing a small-molecule simulated coupling reaction with a toxin linker, this application can obtain a toxin linker-small-molecule conjugate (the first conjugate) that is similar to the actual coupling state. This conjugate is then combined with liquid chromatography, and data is acquired using a dual-wavelength UV detector. Since chromatography can effectively separate the target conjugate from impurities, no additional purification steps are required; the peak areas of the target conjugate at the two wavelengths can be directly analyzed, and the ratio k of the absorbance levels can be calculated.

[0085] The detection method of this application eliminates the limitations of traditional UV methods in handling changes in UV absorption characteristics after coupling, expands the application scope of UV methods in ADC protein content determination, improves detection accuracy, and provides an efficient and accurate detection method for antibody protein content detection for ADC drug research and development, production and quality control, providing technical support for the precise preparation and clinical application of ADC drugs.

[0086] In a preferred embodiment, in a2), the molar ratio of the toxin linker to the coupling molecule is 1:5 to 20, including but not limited to 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, more preferably 1:10.

[0087] When preparing the solution of the first conjugate, an excess of the conjugate molecule can be added to the toxin linker solution to ensure sufficient reaction between the toxin linker and the conjugate molecule. Those skilled in the art can flexibly adjust the mixing ratio of the toxin linker and the conjugate molecule according to the actual system. In a preferred embodiment, the chromatography includes one or more of the following: reversed-phase chromatography (RP), normal-phase chromatography (NP), hydrophilic-interaction chromatography (HILIC), hydrophobic chromatography (HIC), ion-exchange chromatography (IEC), or capillary electrophoresis (CE).

[0088] This application employs suitable liquid chromatography (LC) or capillary electrophoresis (CE) to separate the toxin linker solution from other impurities, obtaining a highly accurate absorbance ratio k. The liquid chromatography methods used include, but are not limited to, reverse-phase chromatography (RP), normal-phase chromatography (NP), hydrophobic interaction chromatography (HIC), and hydrophilic interaction liquid chromatography (HILIC), as well as charge-interaction-based ion exchange chromatography (IEC), all of which can achieve the technical effects described in this application. Those skilled in the art can flexibly select the appropriate method based on actual operation and requirements.

[0089] In a preferred embodiment, the method for determining the molar extinction coefficient of the antibody protein includes: ultraviolet spectrophotometry, amino acid sequence analysis, SEC-UV-RI-MALS, or denaturation treatment.

[0090] The extinction coefficient of antibody protein can be easily obtained through theoretical calculation or actual measurement of pure antibody protein. Those skilled in the art can flexibly choose any of the existing methods for determining the extinction coefficient of antibody protein according to actual needs, and obtain the extinction coefficient of antibody protein.

[0091] In a preferred embodiment, ultraviolet spectrophotometry includes preparing an antibody protein solution of known concentration, scanning the absorption spectrum, and calculating the extinction coefficient of the antibody protein based on the absorbance at a specific wavelength, the antibody concentration, and the optical path length.

[0092] Amino acid sequence analysis includes calculating the extinction coefficient of the antibody protein at a wavelength of 280 nm based on the sequence of the antibody protein.

[0093] The basic principle of the SEC-UV-RI-MALS method is to separate antibodies using size exclusion chromatography (SEC), and then simultaneously detect the eluted antibody proteins using an ultraviolet (UV) detector, a refractive index (RI) detector, and a multi-angle laser light scattering (MALS) detector. The extinction coefficient of the antibody protein is calculated based on the absorbance measured by the UV detector and the molecular weight measured by the MALS detector.

[0094] The basic principle of denaturation treatment is to denature the antibody using reagents such as guanidine hydrochloride, opening up the higher-order structure of the antibody protein folding, treating it as a ternary system of tryptophan, tyrosine and "SS" bonds, and calculating the extinction coefficient of the antibody protein by detecting the absorbance of folded and unfolded antibodies at a wavelength of 280 nm in the ultraviolet light at the same concentration.

[0095] This application overcomes the technical bottleneck of traditional UV methods for protein content determination by combining liquid chromatography separation and dual-wavelength detection, reducing interference from toxin linker purity. Furthermore, this application introduces a small-molecule simulated coupling reaction, solving the problem of changes in UV absorption characteristics after coupling. The fusion of the small-molecule simulated coupling reaction and liquid chromatography separation technology in this application avoids the adverse effects of impurities in the toxin linker on the measurement results. Even when the UV absorption characteristics of the toxin linker change significantly after coupling with the antibody, the UV method can still accurately determine the protein content of the ADC. The detection method of this application expands the application boundaries of UV absorbance methods in ADC drug analysis, improves the accuracy and reliability of the determination, and provides a more efficient and stable technical means for the research, development, production, and quality control of ADC drugs.

[0096] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0097] Example 1

[0098] 1. In this embodiment, the toxin linker is a phenyl isothiocyanate small molecule, wherein the phenyl isothiocyanate functional group undergoes a coupling reaction with the primary amine in the lysine residue of the antibody, as shown in the schematic diagram below. Figure 1 As shown, when the toxin linker couples with the antibody, the phenyl isothiocyanate group is converted into a phenylthiourea group, resulting in a significant change in the absorption spectrum.

[0099] By replacing the lysine residues on the antibody with 6-aminohexanoic acid, the reaction with the toxin linker under the above antibody-toxin conjugation conditions was simulated. A schematic diagram of the reaction is shown below. Figure 2 As shown, after the reaction, a structure similar to that of the toxin linker coupled to the antibody is formed.

[0100] The steps are as follows:

[0101] 1) Prepare a low-purity toxin linker solution with a concentration of approximately 1 mg / mL using a mixture of sodium bicarbonate buffer (pH > 8) and DMSO solvent.

[0102] 2) Add 6-aminohexanoic acid to the toxin linker solution at a molar ratio of 1:10, and shake at room temperature in the dark for about 24 hours to allow the toxin linker and 6-aminohexanoic acid to react fully, thus obtaining a solution of the first conjugate.

[0103] 3) Transfer the unreacted toxin linker original solution and the solution after reaction with 6-aminocaproic acid (the solution of the first conjugate) to liquid chromatography vials respectively.

[0104] 4) Connect a C18 reversed-phase column to the liquid chromatograph, use water containing 0.1% TFA and acetonitrile containing 0.1% TFA as the mobile phase, set a gradient elution program (the acetonitrile ratio increases from 5% to 40% within 10 minutes), set 250 nm and 280 nm as the detection wavelengths for liquid chromatography, and simultaneously set the diode array detector (DAD) to perform a full wavelength scan in the range of 200 nm to 400 nm.

[0105] 5) After equilibrating the column, perform chromatographic analysis on the unreacted toxin linker solution and the solution after reaction with 6-aminocaproic acid. Obtain chromatograms at 250 nm and 280 nm, and simultaneously obtain the absorption spectra of the chromatographic peaks of the toxin linker before and after the reaction with 6-aminocaproic acid.

[0106] 2. The ultraviolet absorption spectra of the free toxin linker and the toxin linker-6-aminohexanoic acid conjugate (the first conjugate) were determined by diode array detector (DAD).

[0107] It is evident that the absorption spectrum of the product after the reaction of the toxin linker with 6-aminohexanoic acid changes significantly compared to the initial reaction, such as... Figure 3 As shown, where, Figure 3 Image A is the UV absorption spectrum of the toxin linker-6-aminohexanoic acid conjugate (the first conjugate). Figure 3 In Figure B, the ultraviolet absorption spectrum of the toxin linker is shown. If the extinction coefficient of the free toxin linker is used directly to calculate the ADC protein content, it will lead to incorrect results.

[0108] Direct reversed-phase chromatography analysis of the reaction solution after the reaction of the toxin linker with 6-aminohexanoic acid can separate the toxin linker-aminohexanoic acid conjugate from other impurities. A UV detector was set to scan the chromatogram of the toxin linker-aminohexanoic acid conjugate (first conjugate) at two wavelengths of 250 nm and 280 nm. Figure 4 As shown, where, Figure 4 Chromatogram A is the chromatogram of the toxin linker aminocaproic acid conjugate (first conjugate) detected at a wavelength of 250 nm; Figure 4 Chromatogram B is the chromatogram of the toxin linker aminocaproic acid conjugate (first conjugate) detected at a wavelength of 280 nm.

[0109] The chromatographic peak of the toxin linker aminocaproic acid conjugate (first conjugate) was integrated, and the ratio of its peak area at two wavelengths was calculated. This yielded the k value in the protein content calculation formula. The k value was then substituted into the formula. The antibody protein content in the ADC sample was obtained.

[0110] The ADC protein content measured according to the method of this application is consistent with the prediction result of the lysine coupling process (within ±10%). If the protein content is detected by the traditional UV dual-wavelength method, the result is more than 30% lower than the prediction result of the lysine coupling process.

[0111] Example 2

[0112] 1. In this embodiment, the toxin linker is a camptothecin molecule containing a sulfone-based heterocyclic aromatic hydrocarbon. The sulfone-based heterocyclic aromatic hydrocarbon functional group therein undergoes a coupling reaction with the free thiol group of cysteine ​​in the antibody. The reaction diagram is shown below. Figure 5 As shown, after the coupling reaction between the toxin linker and the antibody occurs, the methyl sulfone group is replaced by the free thiol group on the antibody, and the absorption spectrum of the coupled toxin linker will change significantly.

[0113] By replacing cysteine ​​residues on the antibody with acetylcysteine, the reaction with the toxin linker under the above antibody-toxin conjugation conditions was simulated. A schematic diagram of the reaction is shown below. Figure 6 As shown, after the reaction, a structure similar to that of the toxin linker coupled to the antibody is formed.

[0114] The steps are as follows:

[0115] 1) Prepare a toxin linker solution with a concentration of approximately 1 mg / mL using a mixture of neutral (pH 7) sodium phosphate buffer and DMSO solvent;

[0116] 2) Add acetylcysteine ​​to the toxin linker solution at a molar ratio of 1:10, and shake at room temperature in the dark for 1 hour to allow the toxin linker and acetylcysteine ​​to react and obtain the solution of the first conjugate.

[0117] 3) Transfer the unreacted toxin linker and the toxin linker reaction solution after reaction with acetylcysteine ​​(the solution of the first conjugate) to two liquid chromatography vials respectively.

[0118] 4) Connect a C18 reversed-phase column to the liquid chromatograph, use water containing 0.1% TFA and acetonitrile containing 0.1% TFA as the mobile phase, set a gradient elution program (the acetonitrile ratio increases from 20% to 70% within 10 minutes), set 260 nm and 280 nm as the detection wavelengths for liquid chromatography, and simultaneously set the diode array detector (DAD) to perform a full wavelength scan in the range of 200 nm to 400 nm.

[0119] 5) After equilibrating the column, perform chromatographic analysis on the unreacted toxin linker solution and the toxin linker solution after reaction with acetylcysteine. Obtain chromatograms at 260 nm and 280 nm, and simultaneously obtain the absorption spectra of the chromatographic peaks of the toxin linker before and after the reaction with acetylcysteine.

[0120] 2. The UV absorption spectra of the free toxin linker and the toxin linker-acetylcysteine ​​conjugate (first conjugate) were measured using a diode array detector (DAD). It was observed that the absorption spectrum of the product after the reaction of the toxin linker with acetylcysteine ​​changed significantly compared to before the reaction. Figure 7 As shown, where, Figure 7 Image A shows the UV absorption spectrum of the toxin linker-acetylcysteine ​​conjugate (the first conjugate). Figure 7 In Figure B, the ultraviolet absorption spectrum of the toxin linker is shown. If the extinction coefficient of the free toxin linker is used directly to calculate the ADC protein content, it will lead to incorrect results.

[0121] Direct reversed-phase chromatography analysis of the reaction solution after the reaction of the toxin linker and acetylcysteine ​​can separate the toxin linker-acetylcysteine ​​conjugate from other impurities. A UV detector is set to scan the chromatogram of the toxin linker-acetylcysteine ​​conjugate (first conjugate) at two wavelengths of 260 nm and 280 nm. Figure 8 As shown, where, Figure 8 Chromatogram A is the chromatogram of the toxin linker-acetylcysteine ​​conjugate (first conjugate) detected at a wavelength of 260 nm. Figure 8 Chromatogram B is the chromatogram of the toxin linker-acetylcysteine ​​conjugate (first conjugate) detected at a wavelength of 280 nm.

[0122] The chromatographic peaks of the toxin linker-acetylcysteine ​​conjugate were integrated, and the peak area ratio at the two wavelengths was calculated to obtain the k value in the protein content calculation formula. This k value was then substituted into the formula. The antibody protein content in the ADC sample was obtained.

[0123] The ADC protein content measured according to the method of this application is consistent with the prediction results of the cysteine ​​coupling process (within ±10%). If the protein content is detected by the traditional UV dual-wavelength method, the result is more than 20% higher than the prediction results of the cysteine ​​coupling process.

[0124] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The detection method of this application does not require high purity of the toxin linker. Through the synergistic effect of liquid chromatography separation and dual-wavelength detection, the extinction coefficient ratio of the toxin linker can be obtained, further improving the accuracy of antibody protein content detection results in ADCs. Furthermore, the present invention overcomes the negative impact of changes in the UV absorption characteristics of the toxin linker after the coupling reaction on protein content determination. Relying on the small molecule simulated coupling reaction of this application, combined with liquid chromatography technology, the applicability and accuracy of UV method for determining ADC protein content are improved. The detection method of this application simplifies the ADC analysis process, improves the efficiency of protein content analysis in the ADC R&D and production process, and opens up new pathways for the precise preparation and quality control of ADC drugs.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting the content of an antibody protein in an antibody conjugate drug, characterized by, The antibody conjugate drug comprises an antibody protein and a toxin linker; The detection method comprises: detecting chromatographic peaks of the toxin linker at wavelength 1 and wavelength 2 by using chromatography, and calculating a ratio of areas of the two chromatographic peaks of the toxin linker at wavelength 1 and wavelength 2; The content of the antibody protein is obtained according to the ratio; The wavelength 1 is a characteristic absorption wavelength of the antibody conjugate drug, the antibody protein or the toxin linker between 200 nm and 300 nm; correspondingly, the wavelength 2 is a characteristic absorption wavelength of the antibody conjugate drug, the antibody protein or the toxin linker between 250 nm and 400 nm; The wavelength 1 and the wavelength 2 are different in value; The content of the antibody protein is obtained according to the following formula: ； The subscripts w1 and w2 respectively represent two wavelengths; Wherein, k represents the ratio of areas of the two chromatographic peaks of the toxin linker at the wavelength 1 and the wavelength 2; A w1*ADC , A w2*ADC respectively the absorbance of the antibody conjugate drug at the wavelength 1 and the wavelength 2; and denotes the molar extinction coefficient of the antibody protein at said wavelength 1 and said wavelength 2; C mAb The content of the antibody protein in the antibody conjugate drug, i.e., the molar concentration; L is the optical path of the cuvette; DF is the dilution factor.

2. The detection method according to claim 1, characterized in that, The detection method comprises: S1) dissolving the toxin linker in a solvent to obtain a solution of the toxin linker; S2) injecting the solution of the toxin linker into the chromatograph, separating the toxin linker in the solution of the toxin linker from impurities, obtaining two chromatographic peak diagrams of the toxin linker at the wavelength 1 and the wavelength 2, and calculating the ratio of areas of the chromatographic peaks of the two chromatographic peak diagrams; S3) substituting the ratio, the absorbance of the antibody conjugate drug at the wavelength 1 and the wavelength 2, and the molar extinction coefficient of the antibody protein at the wavelength 1 and the wavelength 2 into the formula of claim 1 to calculate the content of the antibody protein in the antibody conjugate drug.

3. The method of claim 1, wherein The detection method further comprises: a1) dissolving the toxin linker in a solvent to obtain a solution of the toxin linker; a2) mixing the solution of the toxin linker with a conjugate molecule to perform connection, to obtain a solution of a first conjugate; a3) injecting the solution of the first conjugate into the chromatograph, separating the first conjugate in the solution of the first conjugate from impurities, obtaining two chromatographic peak diagrams of the first conjugate at the wavelength 1 and the wavelength 2, and calculating the ratio of areas of the chromatographic peaks of the two chromatographic peak diagrams; a4) substituting the ratio, the absorbance of the antibody conjugate drug at the wavelength 1 and the wavelength 2, and the molar extinction coefficient of the antibody protein at the wavelength 1 and the wavelength 2 into the formula of claim 1 to calculate the content of the antibody protein in the antibody conjugate drug.

4. The detection method according to claim 3, characterized in that, The conjugate molecule comprises an amino-containing compound and / or a sulfhydryl-containing compound.

5. The detection method according to claim 4, characterized in that, The amino-containing compound comprises any one or more of the following: i) a compound containing an amino group and a carboxyl group; ii) an amine compound.

6. The detection method according to claim 5, characterized in that, The compound containing an amino group and a carboxyl group comprises an amino acid and / or an amino caproic acid; The amine compound comprises methylamine and / or ethylamine.

7. The detection method according to claim 6, characterized in that, The sulfhydryl-containing compound comprises one or more of acetylcysteine, cysteine or glutathione.

8. The detection method according to claim 3, characterized in that, In the a2), the molar ratio of the toxin linker mixed with the coupling molecule is 1:5-20.

9. The detection method according to claim 1, characterized in that, The chromatography includes one or more of reverse phase chromatography, normal phase chromatography, hydrophilic interaction chromatography, hydrophobic chromatography, ion exchange chromatography, or capillary electrophoresis.

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