Method for detecting antibody titer

Abstract

The invention provides a method for detecting antibody titer. The detection method comprises the following steps: loading a standard protein sample to an affinity chromatography column, carrying out ultraviolet detection on an elution product of the standard protein while eluting to obtain a standard protein elution curve, and establishing a standard curve of peak area and titer of the elution curve of the standard protein; loading a to-be-detected sample on the affinity chromatography column, performing ultraviolet detection on an elution product of the to-be-detected sample while eluting, and obtaining the antibody titer of the to-be-detected sample by utilizing the standard curve and the elution curve peak area of the to-be-detected sample; wherein the filler of the affinity chromatography column is protein A, a mobile phase A and a mobile phase B are used for elution, and both the mobile phase A and the mobile phase B contain arginine. The mobile phase containing arginine is used for elution of protein A affinity chromatography, the arginine can influence the surface tension of protein or the solubility of amino acid, and the sensitivity and accuracy of antibody titer detection are improved by influencing the hydrophobicity of a sample.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular, to a method for detecting antibody titer. Background Art

[0002] In recent years, with the rapid development of the global biopharmaceutical industry, the market scale of the antibody industry has increased accordingly. Protein A is a protein on the cell wall of Staphylococcus aureus, which can specifically bind to immunoglobulins of humans and various mammals, especially the Fc region of IgG. Affinity chromatography of Protein A is the main method for detecting the antibody titer of antibodies with Fc fragments during cell culture. However, the components in the cell culture supernatant are complex, including impurities such as supplements added during the culture process, cell debris, and host cell proteins. Some antibodies also have a large number of aggregates and fragments, resulting in double peaks or split peaks during the Protein A affinity chromatography analysis, thus affecting the detection results of antibody titer.

[0003] To solve the problem of double peaks or split peaks during antibody titer detection, existing methods include: adding hydrophobic electrolytes (such as tetramethylammonium chloride, tetraethylammonium chloride) to the mobile phase, adding 0.5%-1% Tween, or increasing the buffer concentration to 1.0-2.0 M. There are also cases where the detection anomaly is alleviated by reducing the elution pH. However, the above methods have defects such as toxicity, flammability (adding hydrophobic electrolytes or adding Tween), and instability (increasing the buffer concentration and reducing the elution pH), and cannot be stably utilized for a long time. Therefore, how to provide a simple and effective method for detecting antibody titer that avoids double peaks or split peaks during affinity chromatography analysis is of great significance for the accuracy of antibody titer detection. Summary of the Invention

[0004] The main object of the present invention is to provide a method for detecting antibody titer to solve the problem of double peaks or split peaks in the process of detecting antibody titer in the prior art.

[0005] To achieve the above object, according to the first aspect of the present invention, there is provided a method for detecting antibody titer, the detection method comprising: loading a standard protein onto an affinity chromatography column, while eluting, performing ultraviolet detection on the elution product of the standard protein to obtain a standard protein elution curve, and establishing a standard curve of the peak area of the standard protein elution curve and the titer; loading a test sample onto the affinity chromatography column, while eluting, performing ultraviolet detection on the elution product of the test sample, and using the standard curve and the peak area of the elution curve of the test sample to obtain the antibody titer of the test sample; wherein, the packing material of the affinity chromatography column is Protein A, and elution is performed using mobile phase A and mobile phase B, and both mobile phase A and mobile phase B contain arginine.

[0006] Further, mobile phase A comprises: 45 - 55 mM Tris-HAc, 140 - 160 mM NaCl, and 0.45 - 0.55 M arginine; preferably, mobile phase B comprises: 110 - 130 mM HAc and 0.45 - 0.55 M arginine.

[0007] Further, mobile phase A comprises: 50 mM Tris-HAc, 150 mM NaCl, and 0.5 M arginine.

[0008] Further, mobile phase B comprises: 120 mM HAc and 0.5 M arginine.

[0009] Further, the pH value of mobile phase A is 6.8 - 7.2.

[0010] Further, the pH value of mobile phase B is 2.9 - 3.3.

[0011] Further, elution includes: at 0 - 1 min of elution, equilibration is carried out with mobile phase A; at 1 - 3 min of elution, protein elution is carried out with mobile phase B; at 3 - 6 min of elution, rinsing is carried out with mobile phase A.

[0012] Further, loading the standard product onto the affinity chromatography column includes: diluting the standard protein with a buffer solution in a gradient concentration to obtain 7 - 8 standard proteins with different concentrations, respectively loading them onto the affinity chromatography column, and respectively carrying out elution and UV detection.

[0013] Further, the concentration range of the diluted standard protein is 0.1 - 10 g / L.

[0014] Further, before loading or elution, the standard protein, the sample to be tested, mobile phase A, and mobile phase B are respectively filtered using a 0.22 μm filter.

[0015] Applying the technical solution of the present invention, by using a mobile phase containing arginine for elution in protein A affinity chromatography, arginine can affect the protein surface tension or amino acid solubility, and by affecting the hydrophobicity of the sample, the sensitivity and accuracy of antibody titer detection are improved. This method not only simplifies the experimental process, but also the obtained antibodies have more stable properties, which can ensure the subsequent normal application of the antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0017] Figure 1Shows the UV detection curve of the elution product obtained by performing affinity chromatography to detect antibody titer in the prior art;

[0018] Figure 2 Shows the UV detection curve of the elution product obtained by performing affinity chromatography to detect antibody titer in Example 1;

[0019] Figure 3 Shows the standard curve obtained using the standard protein in Example 1. Detailed implementation manners

[0020] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0021] Glossary:

[0022] Antibody titer: The antibody titer in this application is for the titer detection of antibodies used in biopharmaceutical cell culture, and can also be understood as the concentration of antibodies in the cell culture medium, rather than the antibody level of a biological individual.

[0023] As mentioned in the background art, during the process of detecting the titer of antibody proteins using protein A chromatography, there are situations such as impurity introduction, antibody aggregation or fragmentation, resulting in the peak of the elution product not being a single peak, but a double peak or split peak phenomenon, making the elution result untrustworthy. Existing solutions to the double peak or split peak phenomenon have defects that the reagents used have certain toxicity or will affect the stability of antibody proteins. Therefore, this application aims to provide a method for detecting antibody titer using affinity chromatography that can avoid the double peak or split peak phenomenon and is more stable.

[0024] In the first typical embodiment of this application, a method for detecting antibody titer is provided. The detection method includes: loading the standard protein onto an affinity chromatography column, while eluting, performing UV detection on the elution product of the standard protein to obtain the standard protein elution curve, and establishing a standard curve of the peak area of the standard protein elution curve and the titer; loading the sample to be tested onto the affinity chromatography column, while eluting, performing UV detection on the elution product of the sample to be tested, and using the standard curve and the peak area of the elution curve of the sample to be tested to obtain the antibody titer of the sample to be tested; wherein, the packing material of the affinity chromatography column is protein A, and elution is performed using mobile phase A and mobile phase B, and both mobile phase A and mobile phase B contain arginine.

[0025] In the elution stage of protein A affinity chromatography in this application, a mobile phase containing arginine is used. Arginine can affect the surface tension of proteins or the solubility of amino acids. An interaction can also occur between the guanidyl group of arginine and the side chain of tryptophan. By affecting the hydrophobicity of the sample, the resolution of analytical detection is improved to obtain a clear single peak, effectively enhancing the sensitivity of antibody titer detection. In the field of biopharmaceuticals, this method can be used to rapidly evaluate the purity and activity of antibody drugs, providing key data support for drug research and development. Among them, the standard protein and the sample to be tested include antibodies with an Fc domain.

[0026] Any buffer solution that can keep the antibody stable in the protein A affinity chromatography system is applicable to this application. In a preferred embodiment, mobile phase A includes: Tris-HAc, NaCl, and arginine; preferably, mobile phase B includes: HAc and arginine. Preferably, mobile phase A includes: 50 mM Tris-HAc, 150 mM NaCl, and 0.5 M arginine. Preferably, mobile phase B includes: 120 mM HAc and 0.5 M arginine. Preferably, the pH value of mobile phase A is 7.0. Preferably, the pH value of mobile phase B is 3.1.

[0027] The binding of protein A to the Fc segment of the antibody is mainly based on the electrostatic interaction between His residues. By gradually decreasing the pH value of the mobile phase, the antibody is dissociated from protein A and eluted. In order to elute the antibody without destroying its properties, in a preferred embodiment, the elution includes: in the 0 - 1 min of elution, balance with mobile phase A; in the 1 - 3 min of elution, elute the protein with mobile phase B; in the 3 - 6 min of elution, wash with mobile phase A. Among them, in the balance stage, reagents and related impurities other than the antibody are eluted; in the protein elution stage, the antibody is eluted from protein A, and the peak in this stage is the eluted antibody. Subsequently, the affinity chromatography column is washed and then regenerated and stored. In the washing stage, mobile phase A is used to replace mobile phase B with a lower pH value relatively completely, and the detection system is washed to a neutral condition. Moreover, due to the large volume of the detection system, it is necessary to further ensure that the sample in the detection system has been completely rinsed off to avoid affecting the detection results of the next sample.

[0028] In order to obtain a more accurate standard curve, the standard protein is diluted into multiple different gradients for detection. In a preferred embodiment, loading the standard protein onto the affinity chromatography column includes: diluting the standard protein with a buffer solution to obtain 7 - 8 standard proteins with different concentrations, respectively loading them onto the affinity chromatography column, and respectively performing elution and ultraviolet detection.

[0029] The concentration gradient range of the standard protein may be set according to the concentration of the antibody to be detected. In a preferred embodiment, the concentration range of the diluted standard protein is 0.1 - 10 g / L.

[0030] In order to further filter out some impurities, in a preferred embodiment, before sample loading or elution, the standard protein, the sample to be tested, mobile phase A, and mobile phase B are respectively filtered using a 0.22 μm filter.

[0031] The following further describes the present application in detail with specific embodiments, which should not be construed as limiting the scope claimed by the present application.

[0032] Example 1

[0033] 1. Preparation before detection

[0034] 1.1 Sample preparation: Gradient concentration dilution of rituximab (standard protein) is performed using ultrapure water. The concentration of the standard protein is detected using an ultraviolet spectrophotometer, and it is diluted step by step from high concentration to low concentration to prepare standard proteins with 8 concentration ranges, which are 10 g / L, 5.0 g / L, 2.5 g / L, 1.25 g / L, 0.63 g / L, 0.31 g / L, 0.15 g / L, and 0.1 g / L respectively. The standard protein solution with gradient concentration is filtered using a 0.22 μm filter. (Please note that the concentration gradient is not fixed in actual use, and a curve with good linearity and covering the concentration range to be detected can be obtained.)

[0035] Mobile phase A: 50 mM Tris-HAc, 150 mM NaCl, 0.5 M Arg, pH 7.0;

[0036] Mobile phase B: 120 mM HAc, 0.5 M Arg, pH 3.1.

[0037] The prepared mobile phase A and B, and the standard protein are filtered using a 0.22 μm filter and degassed by ultrasonic treatment.

[0038] Column packing material: ChromaX A20 (BioLink).

[0039] 1.2 System flushing: Flush all mobile phase pipelines with buffer solution, balance the chromatography column with buffer solution, and balance the system to a stable baseline at a flow rate of 1.0 mL / min with the initial mobile phase ratio.

[0040] 2. Sample detection

[0041] First, affinity chromatography is performed on standard proteins with different concentrations in sequence from high concentration to low concentration.

[0042] The specific affinity chromatography method is as follows: Load the standard protein onto the affinity chromatography column, and perform elution and UV detection. The elution process is divided into three stages: During the elution from 0 to 1 minute, perform the first elution using mobile phase A; during the elution from 1 to 3 minutes, perform the second elution using mobile phase B; during the elution from 3 to 6 minutes, perform the third elution using mobile phase A to complete the elution. Record the UV detection while eluting (wherein, the top layer of the HPLC detection instrument is the UV detector, and the UV spectrum can be obtained simultaneously).

[0043] After integrating the elution peaks of the standard proteins at the above different concentrations, establish a standard curve with the peak area as the abscissa and the concentration as the ordinate (as Figure 3 shown). Then perform the above affinity chromatography operation on the sample to be detected (i.e., the prepared standard protein with an unknown concentration) to obtain the corresponding peak area (as Figure 1 shown), wherein, Figure 1 the second peak is the elution peak of the protein. Substitute it into the regression equation to calculate that the concentration of the sample to be detected is 3.16 g / L.

[0044] Comparative Example 1

[0045] In this comparative example, except that the types of mobile phases A and B for elution and the affinity chromatography elution process are different from those in Example 1, the remaining operations are the same as those in Example 1. The compositions of mobile phases A and B in this comparative example are as follows:

[0046] Mobile phase A: 50 mM Tris-HAc, 150 mM NaCl, pH 7.0;

[0047] Mobile phase B: 120 mM HAc.

[0048] The specific affinity chromatography process is as follows: Load the sample to be detected onto the affinity chromatography column, and perform elution and UV detection. The elution process is divided into three stages: Start loading at 0 minute, and perform top washing using mobile phase A from 0 to 2 minutes after loading; from 2 to 4 minutes after loading, elute the sample using mobile phase B; from 3 to 6 minutes during elution, perform equilibration and top washing using mobile phase A to complete the detection, and obtain the protein elution peak as shown in Figure 1 shown. It can be seen that Figure 2 the second peak is not a single peak, and the accurate protein concentration value cannot be calculated.

[0049] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: By using a mobile phase containing arginine for elution in protein A affinity chromatography, the antibody protein bound to protein A can be more specifically and thoroughly separated, improving the sensitivity and accuracy of antibody titer detection. This method not only simplifies the experimental process, but also the obtained antibodies have more stable properties, ensuring the subsequent normal application of antibodies. It provides an efficient and reliable solution for antibody detection, especially suitable for the rapid screening of large-scale antibody titers, and has significant application value in the fields of biopharmaceuticals, clinical diagnosis, etc.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting antibody titer, characterized in that, The detection method includes: Loading the standard protein onto an affinity chromatography column, while eluting, performing ultraviolet detection on the elution product of the standard protein to obtain an elution curve of the standard protein, and establishing a standard curve of the peak area of the elution curve of the standard protein and the titer. Loading the sample to be tested onto the affinity chromatography column, while eluting, performing the ultraviolet detection on the elution product of the sample to be tested, and obtaining the antibody titer of the sample to be tested by using the standard curve and the peak area of the elution curve of the sample to be tested. Among them, the packing material of the affinity chromatography column is Protein A, and the elution is performed by using mobile phase A and mobile phase B, and both mobile phase A and mobile phase B contain arginine.

2. The detection method according to claim 1, wherein Mobile phase A includes: 45 - 55 mM Tris-HAc, 140 - 160 mM NaCl, and 0.45 - 0.55 M arginine. Preferably, mobile phase B includes: 110 - 130 mM HAc and 0.45 - 0.55 M arginine.

3. The detection method according to claim 2, characterized in that, Mobile phase A includes: 50 mM Tris-HAc, 150 mM NaCl, and 0.5 M arginine.

4. The detection method according to claim 2, characterized in that, Mobile phase B includes: 120 mM HAc and 0.5 M arginine.

5. The detection method according to claim 2, wherein The pH value of mobile phase A is 6.8 - 7.

2.

6. The detection method according to claim 2, characterized in that The pH value of mobile phase B is 2.9 - 3.

3.

7. The detection method according to claim 1, wherein The elution includes: at 0 - 1 min of elution, equilibration is performed by using mobile phase A; at 1 - 3 min of elution, protein elution is performed by using mobile phase B; at 3 - 6 min of elution, rinsing is performed by using mobile phase A.

8. The detection method according to claim 1, characterized in that, The loading of the standard onto the affinity chromatography column includes: Gradiently diluting the standard protein with a buffer solution to obtain 7 - 8 standard proteins with different concentrations, respectively loading them onto the affinity chromatography column, and respectively performing the elution and the ultraviolet detection.

9. The detection method according to claim 8, wherein The concentration range of the diluted standard protein is 0.1 - 10 g / L.

10. The detection method according to claim 1, wherein Before performing the loading or the elution, the standard protein, the sample to be tested, mobile phase A, and mobile phase B are respectively filtered by using a 0.22 μm filter.

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