Method for detecting cationic chromatography dynamic capacity under influence of small molecules, applications

Abstract

The application discloses a method and application for detecting dynamic load of cation chromatography affected by small molecules, which comprises the following steps: (1) after balancing the cation chromatography column, a reaction solution containing small molecules is used for sample loading until the A280 absorption value reaches a platform; (2) the flow-through sample is collected in sections, and the corresponding sample loading amount is recorded; (3) the sample loading and the flow-through sample are subjected to SEC liquid chromatography detection in equal volume, and the main peak area of each sample is obtained; (4) the ratio of the main peak area of each flow-through sample to the SEC liquid chromatography is calculated; (5) the load-flow-through sample SEC liquid chromatography main peak area ratio curve is fitted according to the ratio; and (6) the corresponding load of the product flow-through amount of 10% is calculated according to the fitted curve. Advantages are as follows: (1) the small molecule impurities are separated and quantified by combining SEC liquid chromatography and fitting model, and the maximum dynamic binding load of the sample loading on the chromatography column filler is calculated through the model curve; (2) the detection sensitivity is high, the accuracy is good, and the application range is wide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemistry, and relates to a method for measuring dynamic binding capacity of cation chromatography, in particular to a method for detecting dynamic binding capacity of cation chromatography affected by small molecules in the production process of antibody conjugated drugs and application. BACKGROUND

[0002] In the production process of antibody conjugated drugs, cation chromatography is a common purification method for ADC reaction liquid. If the free small molecules have ultraviolet absorption, the ultraviolet absorption value of the flow-through liquid of cation chromatography will be directly affected, thereby affecting the measurement of dynamic binding capacity. The traditional method for measuring dynamic binding capacity of cation chromatography is to monitor the A280 absorption value of the flow-through liquid in real time. When the absorption value of the flow-through liquid reaches 10% of the absorption value of the sample, that is, the concentration of the ADC molecules in the flow-through liquid reaches 10% of the concentration of the sample, the capacity corresponding to this time is the maximum dynamic binding capacity of the ADC molecules on the filler. The disadvantage of this method is that if the free small molecules have ultraviolet absorption, the ultraviolet absorption value of the chromatographic flow-through liquid is generated by the small molecules and the ADC molecules in the flow-through liquid, so that the dynamic binding capacity cannot be accurately determined, and the error of the calculated capacity result is large.

[0003] By using the characteristic that SEC liquid chromatography can separate small molecule impurities, the content of the target ADC molecules in the flow-through liquid is quantitatively detected, so that the dynamic binding capacity of cation chromatography can be accurately calculated. However, the detection result obtained by SEC liquid chromatography is not an intuitive data. The peak area ratio of the main peak of the flow-through liquid corresponding to the SEC liquid chromatography needs to be plotted into a curve, and the capacity corresponding to the target flow-through ratio is obtained after fitting the model. SUMMARY

[0004] The technical problem to be solved is to overcome the shortcomings of the prior art and solve the problem of inaccurate dynamic binding capacity of cation chromatography caused by ultraviolet absorption of small molecules. By combining SEC liquid chromatography and model fitting, a method for detecting dynamic binding capacity of cation chromatography affected by small molecules and application are provided.

[0005] Technical scheme: The method for detecting dynamic binding capacity of cation chromatography affected by small molecules comprises the following steps:

[0006] S1, equilibrate the cation chromatography column, set the retention time, and sample with a reaction liquid containing small molecules until the A280 absorption value reaches a platform;

[0007] S2, collect the flow-through samples in sections during the sampling process, and record the sample loading capacity corresponding to each flow-through sample;

[0008] S3, sample the sample in S1 and each flow-through sample collected in S2 by equal volume, and detect by SEC liquid chromatography to obtain the main peak area of each sample;

[0009] S4, respectively calculating the ratio of the peak area of the main peak of SEC liquid chromatography of each flow-through sample to the sample, i.e. the proportion of ADC content in the total protein content;

[0010] S5, fitting the load-flow-through sample SEC liquid chromatography main peak area ratio curve according to the ratio in S4, and the fitting model is a linear model, a two-parameter logistic model or a three-parameter logistic model;

[0011] S6, calculating the corresponding load of the reaction liquid flow-through amount of 10% according to the curve fitted in S5.

[0012] Preferably, in S1, the small molecule in the reaction liquid is a small molecule with ultraviolet absorption characteristics.

[0013] Preferably, in S5, a data statistical tool is used for fitting, wherein,

[0014] The formula of the two-parameter logistic model is y=1 / (1+exp(-a*(x-b))) (corrected formula), x is the sample load, y is the ratio of the peak area of the main peak of SEC liquid chromatography of each flow-through sample to the sample, a is the growth rate, b is the inflection point, and exp is the natural exponential function;

[0015] The formula of the three-parameter logistic model is y=c / (1+exp(-a*(x-b))), x is the sample load, y is the ratio of the peak area of the main peak of SEC liquid chromatography of each flow-through sample to the sample, a, b, and c are parameters of the model, a is the growth rate, b is the inflection point, c is the asymptote, and exp is the natural exponential function.

[0016] The above-mentioned method is used for detecting the dynamic load of the ADC product cationic chromatography affected by the ultraviolet absorption of the small molecule.

[0017] Preferably, the small molecule is a methyl australin E.

[0018] Preferably, the fitted three-parameter logistic model formula is y=0.980 / (1+exp(-0.255*(x-118.94))), wherein x is the sample load, y is the ratio of the peak area of the main peak of SEC liquid chromatography of each flow-through sample to the sample, and exp is the natural exponential function.

[0019] Beneficial effects: (1) The method described in the present application combines SEC liquid chromatography and fitting model, which separates and quantifies small molecule impurities on one hand, and calculates the corresponding load of the reaction liquid flow-through amount of 10% on the other hand, i.e. the maximum dynamic binding load of the sample on the chromatographic column filler; (2) The detection sensitivity of the method is high, the accuracy is good, and the application range is wide. Attached Figure Description

[0020] Figure 1 This is the chromatographic pattern of the dynamic cation loading of the ADC reaction solution determined on a laboratory-scale 2.16 mL Tricorn 5 / 150 column in Example 1.

[0021] Figure 2 This is the SEC-HPLC detection chromatogram from Example 1;

[0022] Figure 3 This is the curve fitted to the ratio of loading to flow strip sample SEC-HPLC main peak area in Example 1;

[0023] Figure 4 Example 2 shows the chromatographic pattern of the dynamic cation loading of the ADC reaction solution determined on a laboratory-scale 6.158 mL 0.66 cm x 20 cm column;

[0024] Figure 5 The curve is the fitting curve of the ratio of loading to flow-through sample SEC-HPLC main peak area in Example 2. Detailed Implementation

[0025] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0026] Example 1 uses a three-parameter logistic model

[0027] This embodiment predicts the dynamic loading capacity of cation exchange chromatography packing material in the antibody-drug conjugate (ADC) reaction solution during the production process. Praeato SP65 packing material and a Tricorn 5 column (11 cm high, 2.16 mL, As = 1.36, HETP: 0.04 cm) were used to prepare flow-through samples. First, SEC-HPLC was used to detect the loading and flow-through of the cation exchange chromatography sample affected by the UV absorption of small molecule esters (MMAE). Then, the dynamic loading capacity of the cation exchange chromatography packing material was predicted using a fitted curve. The specific steps include:

[0028] S1. After equilibrating the cation exchange column, set the retention time to 5 minutes, and load the sample with ADC reaction solution until the A280 absorbance reaches the plateau of 2155 mAU. The sample concentration is 7.0 mg / ml.

[0029] S2. During the sample loading process, 0.25 ml of flow-through sample is collected for every 2.73 ml of sample loaded, i.e., a sample is taken for every 10 mg / ml loading. See Table 1 for the corresponding loading amounts for each sample tube. The chromatograms are shown below. Figure 1 ;

[0030] S3. Load the same volume of the sample and the collected flow-through sample (only samples with UV absorption are detected) for SEC-HPLC analysis. The peak areas of the main peaks for each sample are shown in Table 1, and the SEC-HPLC chromatograms are shown in [Table 1]. Figure 2 ;

[0031] S4. Calculate the ratio of the main peak area of ​​each flow-through sample to that of the loaded sample. The results are detailed in Table 1.

[0032] S5. Fit the loading-flow-through sample SEC-HPLC main peak area ratio curve based on the ratio in S4. The fitting model is shown in [reference]. Figure 3 The formula is y = 0.980 / (1 + expP(-0.255*(x-118.94))), where x is the loading amount, y is the ratio of flow through to the peak area of ​​the main peak of the SEC-HPLC sample, and exp is the natural exponential function.

[0033] S6. Calculate the loading corresponding to 10% flow rate of the ADC product when the retention time is 5 minutes, i.e., y = 0.1, and calculate x = ln(0.980 / 0.1 -1) / (-0.255) + 118.94 = 110.41 mg / ml resin.

[0034] S7. If the traditional dynamic loading determination method of cation chromatography is followed, when the flow-through absorbance reaches 10% of the sample absorption, i.e., 2155mAU*10%=216mAU, the corresponding loading of 112.77mg / ml resin is 102% of the loading in S6.

[0035] Table 1 shows the loading, main peak area, and the ratio of the main peak area of ​​each flow-through sample to the loaded sample in Example 1.

[0036]

[0037] Example 2 uses a two-parameter logistic model

[0038] This embodiment predicts the dynamic loading capacity of cation exchange chromatography packing material in the antibody-drug conjugate (ADC) reaction solution during the production process. Praeato SP65 packing material and a 0.66 cm column (18.0 cm high, 6.158 mL, As = 0.82, HETP: 0.04 cm) were used to prepare flow-through samples. First, SEC-HPLC was used to detect the loading and flow-through of the cation exchange chromatography sample affected by the UV absorption of small molecule esters (MMAE). Then, the dynamic loading capacity of the cation exchange chromatography packing material was predicted using a fitted curve. The specific steps include:

[0039] S1. After equilibrating the cation exchange column, set the retention time to 3 minutes, and load the sample with ADC reaction solution until the A280 absorbance reaches the plateau of 1531 mAU. The sample concentration is 5.9 mg / ml.

[0040] S2. During the sample loading process, 0.83 ml of flow-through sample is collected for every 10.33 ml of sample loaded, i.e., a sample is taken for every 10 mg / ml loading. See Table 2 for the corresponding loading amounts for each sample tube. The chromatograms are shown below. Figure 4 ;

[0041] S3. Load the same volume of the sample and the collected flow-through sample (only samples with UV absorption are detected) onto the sample and perform SEC-HPLC analysis. The peak areas of the main peaks of each sample are shown in Table 2.

[0042] S4. Calculate the ratio of the main peak area of ​​each flow-through sample to that of the loaded sample. The results are detailed in Table 2.

[0043] S5. Fit the loading-flow-through sample SEC-HPLC main peak area ratio curve based on the ratio in S4. The fitting model is shown in [reference]. Figure 3 The formula is c / c0=1 / (1+exp(-0.049*(DBC-133.51))), where DBC is the loading amount, c / c0 is the ratio of flow through to the peak area of ​​the main peak of the SEC-HPLC sample, and exp is the natural exponential function.

[0044] S6. Calculate the loading of the ADC product when the retention time is 3 minutes and the flow rate is 10%, i.e., c / c0 = 0.1, according to the fitting formula in S5. Calculate DBC = ln(1 / 0.1 -1) / (-0.049) + 133.51 = 88.67 mg / ml resin.

[0045] S7. If the traditional dynamic loading determination method of cation chromatography is followed, when the flow-through absorbance reaches 10% of the sample absorption, i.e., 1531mAU*10%=153mAU, the corresponding loading of 11.2mg / ml resin obtained at this time is only 12% of the loading in S6.

[0046] Table 2 shows the loading, main peak area, and the ratio of the main peak area of ​​each flow-through sample to the loaded sample in Example 2.

[0047]

Claims

1. A method for detecting the dynamic loading of cationic chromatography affected by small molecules, characterized in that, The method comprises the following steps: S1, equilibrating a cation chromatography column, setting a retention time, and loading a reaction liquid containing small molecules until the A280 absorption value reaches a platform; the small molecules in the reaction liquid have ultraviolet absorption characteristics; S2, collecting flow-through samples in sections during the loading process and recording the loading capacity corresponding to each flow-through sample; S3, loading the sample in S1 and each flow-through sample collected in S2 in an equal volume for SEC liquid chromatography detection to obtain the main peak area of each sample; S4, calculating the ratio of the SEC liquid chromatography main peak area of each flow-through sample to the sample, i.e., the proportion of the ADC content in the total protein content; S5, fitting the loading-flow-through loading SEC liquid chromatography main peak area ratio curve according to the ratio in S4, and the fitting model is a linear model, a two-parameter logistic model or a three-parameter logistic model; S6, calculating the corresponding loading amount when the flow-through amount of the reaction liquid containing small molecules is 10% according to the curve fitted in S5.

2. The method of claim 1, wherein the cationic chromatography dynamic loading detection method is affected by a small molecule. In S5, data statistical tools are used for fitting, wherein, The formula of the two-parameter logistic model is y=1 / (1+exp(-a*(x-b))), x is the loading capacity, y is the ratio of the SEC liquid chromatography main peak area of each flow-through sample to the sample, a is the growth rate, b is the inflection point, and exp is the natural exponential function; The formula of the three-parameter logistic model is y=c / (1+exp(-a*(x-b))), x is the loading capacity, y is the ratio of the SEC liquid chromatography main peak area of each flow-through sample to the sample, a, b and c are parameters of the model, a is the growth rate, b is the inflection point, c is the asymptote, and exp is the natural exponential function.

3. The application of the small molecule-affected cation chromatography dynamic loading detection method of claim 1 or 2 in detecting the cation chromatography dynamic loading of an antibody-drug conjugate (ADC) product affected by small molecule ultraviolet absorption.

4. Use according to claim 3, characterized in that, The small molecule is a methyl australin E.

5. Use according to claim 3, characterized in that, The fitted three-parameter logistic model formula is y=0.980 / (1+exp(-0.255*(x-118.94))), wherein x is the loading capacity, y is the ratio of the SEC liquid chromatography main peak area of the flow-through sample to the sample, and exp is the natural exponential function.

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