Method for detecting antibody titer
By using a mobile phase modified with arginine for protein A affinity chromatography elution, the problem of double peaks or split peaks in antibody titer detection is solved, improving the sensitivity and accuracy of detection, simplifying the experimental procedure, and making it suitable for biopharmaceutical and clinical diagnostic fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI KAILAIYING BIOTECHNOLOGY DEVELOPMENT CO LTD
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-08
AI Technical Summary
Existing antibody titer detection processes exhibit bimodal or split peak phenomena, affecting the accuracy of test results. Existing solutions also suffer from drawbacks such as toxicity, flammability, and instability.
Protein A affinity chromatography elution was performed using a mobile phase containing arginine. By influencing protein surface tension and amino acid solubility, the detection resolution was improved, and a standard curve was established to accurately calculate the antibody titer.
This technology improves the sensitivity and accuracy of antibody titer detection, simplifies experimental procedures, ensures stable antibody properties, and is suitable for use in biopharmaceutical and clinical diagnostic fields.
Smart Images
Figure CN121994978A_ABST
Abstract
Description
[0001] This application is based on and claims priority to Chinese application CN application number 2025104718090 filed on April 15, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field
[0002] This invention relates to the field of protein purification technology, and more specifically, to a method for detecting antibody titers. Background Technology
[0003] In recent years, the rapid development of the global biopharmaceutical industry has led to a corresponding increase in the market size of the antibody industry. Protein A is a protein found on the cell wall of Staphylococcus aureus that can specifically bind to immunoglobulins in humans and various mammals, especially the Fc site of IgG. Affinity chromatography of Protein A is the primary method for detecting antibody titers containing Fc fragments during cell culture. However, the composition of cell culture supernatant is complex, containing impurities such as feed additives, cell debris, and host cell proteins. Some antibodies may also exhibit double peaks or split peaks during Protein A affinity chromatography due to the presence of large amounts of aggregates and fragments, thus affecting the antibody titer detection results.
[0004] To address the bimodal or splitting peak phenomenon in antibody titer detection, existing methods include adding hydrophobic electrolytes (such as tetramethylamine chloride or tetraethylamine chloride) to the mobile phase, adding 0.5%-1% Tween, or increasing the buffer concentration to 1.0-2.0 M. Some studies have also attempted to alleviate detection anomalies by lowering the elution pH. However, these methods suffer from drawbacks such as toxicity, flammability (due to the addition of hydrophobic electrolytes or Tween), and instability (due to increased buffer concentration and decreased elution pH), making them unsuitable for long-term stable use. Therefore, providing a simple and effective antibody titer detection method that avoids bimodal or splitting peak phenomena during affinity chromatography is of great significance for the accuracy of antibody titer detection. Summary of the Invention
[0005] The main objective of this invention is to provide a method for detecting antibody titers, thereby solving the problem of double peaks or peak splitting in the existing antibody titer detection process.
[0006] To achieve the above objectives, according to a first aspect of the present invention, a method for detecting antibody titers is provided. The method includes: loading a standard protein onto an affinity chromatography column, eluting while simultaneously detecting the elution product of the standard protein under ultraviolet light 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 versus its titer; loading a test sample onto an affinity chromatography column, eluting while simultaneously detecting the elution product of the test sample under ultraviolet light, and obtaining the antibody titer of the test sample using the standard curve and the peak area of the elution curve 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, both of which contain arginine.
[0007] 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.
[0008] Furthermore, mobile phase A comprises: 50 mM Tris-HAc, 150 mM NaCl, and 0.5 M arginine.
[0009] Furthermore, mobile phase B comprises 120 mM HAc and 0.5 M arginine.
[0010] Furthermore, the pH of mobile phase A is 6.8-7.2.
[0011] Furthermore, the pH of mobile phase B is 2.9-3.3.
[0012] Furthermore, the elution process includes: equilibration using mobile phase A during elution of 0-1 min, protein elution using mobile phase B during elution of 1-3 min, and rinsing using mobile phase A during elution of 3-6 min.
[0013] Further, loading the standard onto the affinity chromatography column includes: diluting the standard protein with a gradient concentration using a buffer solution to obtain 7-8 standard proteins of different concentrations, loading them onto the affinity chromatography column respectively, and performing elution and UV detection respectively.
[0014] Furthermore, the concentration range of the diluted standard protein is 0.1-10 g / L.
[0015] Furthermore, before loading or eluting, the standard protein, the test sample, mobile phase A, and mobile phase B are filtered using a 0.22 μm filter, respectively.
[0016] By applying the technical solution of this invention, elution of protein A using a mobile phase containing arginine is performed. Arginine can affect protein surface tension or amino acid solubility, thereby influencing the hydrophobicity of the sample and improving the sensitivity and accuracy of antibody titer detection. This method not only simplifies the experimental procedure but also yields more stable antibodies, ensuring their proper subsequent application. Attached Figure Description
[0017] 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:
[0018] Figure 1 The UV detection curve of the elution product obtained by affinity chromatography to detect antibody titer in Example 1 is shown.
[0019] Figure 2 The UV detection curve of the elution product obtained by affinity chromatography to detect antibody titer in Comparative Example 1 is shown.
[0020] Figure 3 The standard curve obtained using standard protein in Example 1 is shown. Detailed Implementation
[0021] 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 accompanying drawings and embodiments.
[0022] Glossary:
[0023] Antibody titer: The antibody titer in this application refers to 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 an individual organism.
[0024] As mentioned in the background art, during the detection of antibody protein titers using protein A chromatography, impurities may be introduced or antibodies may aggregate or break down, resulting in double peaks or split peaks in the elution products, making the elution results unreliable. Existing solutions to double peaks or split peaks have the drawback of using reagents with certain toxicity or affecting the stability of antibody proteins. Therefore, this application aims to provide a more stable method for antibody titer detection using affinity chromatography that can avoid double peaks or split peaks.
[0025] In a first typical embodiment of this application, a method for detecting antibody titer is provided. The method includes: loading a standard protein onto an affinity chromatography column, eluting while simultaneously detecting the elution product of the standard protein under ultraviolet light to obtain an elution curve of the standard protein, and establishing a standard curve relating the peak area of the elution curve to the titer; loading a test sample onto an affinity chromatography column, eluting while simultaneously detecting the elution product of the test sample under ultraviolet light, and obtaining the antibody titer of the test sample using the standard curve and the peak area of the elution curve 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, both of which contain arginine.
[0026] This application utilizes a mobile phase containing arginine in the protein A affinity chromatography elution stage. Arginine can affect protein surface tension or amino acid solubility, and the guanidinium group of arginine can also interact with the side chain of tryptophan, affecting the hydrophobicity of the sample and improving the resolution of analytical detection to obtain a clear single peak, effectively enhancing the sensitivity of antibody titer detection. In the biopharmaceutical field, this method can be used to rapidly assess the purity and activity of antibody drugs, providing crucial data support for drug development. The standard protein and the test sample include antibodies containing Fc domains.
[0027] Any buffer solution capable of maintaining the stability of the antibody in the protein A affinity chromatography system is suitable for this application. In a preferred embodiment, mobile phase A comprises Tris-HAc, NaCl, and arginine; preferably, mobile phase B comprises HAc and arginine. Preferably, mobile phase A comprises 50 mM Tris-HAc, 150 mM NaCl, and 0.5 M arginine. Preferably, mobile phase B comprises 120 mM HAc and 0.5 M arginine. Preferably, the pH of mobile phase A is 7.0. Preferably, the pH of mobile phase B is 3.1.
[0028] The binding of protein A to the antibody Fc fragment is primarily via electrostatic interactions between His residues. Gradually decreasing the pH of the mobile phase causes the antibody to dissociate from protein A and be eluted. To elute the antibody without damaging its properties, in a preferred embodiment, elution includes: equilibration with mobile phase A for 0-1 min (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 min); protein elution with mobile phase B for 1-3 min; and rinsing with mobile phase A for 3-6 min (including but not limited to 3, 4, 5, or 6 min). During the equilibration phase, reagents and impurities other than the antibody are eluted. During the protein elution phase, the antibody is eluted from protein A; the peak at this stage represents the eluted antibody. The affinity chromatography column is then rinsed for subsequent regeneration and storage. During the rinsing stage, mobile phase A is used to completely replace mobile phase B, which has a lower pH value, to rinse the detection system to neutral conditions. Since the detection system has a large volume, it is necessary to further ensure that the sample in the detection system has been completely rinsed away to avoid affecting the detection results of the next sample.
[0029] 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 onto the affinity chromatography column includes: diluting the standard protein with a buffer solution to obtain 7-8 standard proteins of different concentrations, loading them onto the affinity chromatography column, and performing elution and UV detection respectively.
[0030] 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 (including but not limited to 0.1, 0.15, 0.2, 0.3, 0.31, 0.4, 0.5, 0.6, 0.63, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10 g / L).
[0031] In order to further filter out some impurities, in a preferred embodiment, the standard protein, the test sample, mobile phase A and mobile phase B are filtered using a 0.22 μm filter before loading or elution.
[0032] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.
[0033] Example 1
[0034] 1. Preparation before testing
[0035] 1.1 Sample Preparation: Rituximab (standard protein) was serially diluted with ultrapure water. The concentration of the standard protein was detected using a UV spectrophotometer. The standard protein was serially diluted from high to low concentration to prepare eight concentration ranges: 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. The standard protein solutions of these concentrations were filtered through a 0.22 μm filter. (Note that the concentration gradient is not fixed in practice; it is sufficient to obtain a linear curve that covers the range of concentrations to be measured.)
[0036] Mobile phase A: 50 mM Tris-HAc, 150 mM NaCl, 0.5 M Arg, pH 7.0;
[0037] Mobile phase B: 120 mM HAc, 0.5M Arg, pH 3.1.
[0038] The prepared mobile phases A and B, along with the standard protein, were filtered through a 0.22 μm filter and degassed by sonication.
[0039] Chromatography column packing material: ChromaX A20 (BioLink).
[0040] 1.2 System flushing: Flush all mobile phase tubing with buffer, equilibrate the chromatography column with buffer, and equilibrate the system to baseline stability at a flow rate of 1.0 mL / min, using the initial mobile phase ratio.
[0041] 2. Sample testing
[0042] First, standard proteins of different concentrations were subjected to affinity chromatography sequentially from high to low concentration.
[0043] The specific affinity chromatography method is as follows: The standard protein is loaded onto an affinity chromatography column for elution and UV detection. The elution process consists of three stages: First elution is performed using mobile phase A during the first 0-1 min; second elution is performed using mobile phase B during the second 1-3 min; and third elution is performed using mobile phase A during the third 3-6 min, completing the elution. UV detection and recording are performed simultaneously during elution (the top layer of the HPLC instrument is equipped with a UV detector, allowing for simultaneous acquisition of the UV spectrum).
[0044] After integrating the elution peaks of the standard proteins at different concentrations, a standard curve was constructed with peak area on the x-axis and concentration on the y-axis (e.g., ...). Figure 3(As shown). Then, the sample to be tested (i.e., the prepared standard protein with an unknown concentration) is subjected to the above affinity chromatography operation to obtain the corresponding peak area (e.g., Figure 1 As shown), where, Figure 1 The second peak is the elution peak of the protein. Substituting it into the regression equation, the concentration of the sample to be tested was calculated to be 3.16 g / L.
[0045] Comparative Example 1
[0046] Except for the types of mobile phases A and B used for elution and the affinity chromatography elution process, the operations in this comparative example are the same as in Example 1. The compositions of mobile phases A and B in this comparative example are as follows:
[0047] Mobile phase A: 50 mM Tris-HAc, 150 mM NaCl, pH 7.0;
[0048] Mobile phase B: 120 mM HAc.
[0049] The specific affinity chromatography procedure is as follows: The sample to be tested is loaded onto the affinity chromatography column, followed by elution and UV detection. The elution process consists of three stages: sample loading begins at 0 min; mobile phase A is used for top washing for 0-2 min after loading; mobile phase B is used for sample elution for 2-4 min after loading; and mobile phase A is used for equilibration and top washing for 3-6 min after elution, completing the detection and obtaining the sample as shown below. Figure 2 The protein elution peaks shown indicate that... Figure 2 The second peak is not a single peak, making it impossible to calculate an accurate protein concentration value.
[0050] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: by using a mobile phase containing arginine for protein A affinity chromatography elution, the antibody protein bound to protein A can be separated more specifically and thoroughly, improving the sensitivity and accuracy of antibody titer detection. This method not only simplifies the experimental procedure, but also yields more stable antibodies, ensuring their proper subsequent application. It provides an efficient and reliable solution for antibody detection, particularly suitable for rapid screening of large-scale antibody titers, and has significant application value in fields such as biopharmaceuticals and clinical diagnostics.
[0051] 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 antibody titers, characterized in that, The detection method includes: The standard protein was loaded onto an affinity chromatography column, and the elution product of the standard protein was detected by UV light while eluting to obtain the elution curve of the standard protein. A standard curve of peak area and titer of the elution curve of the standard protein was established. The sample to be tested is loaded onto the affinity chromatography column, and the elution product of the sample to be tested is subjected to ultraviolet detection while eluting. The antibody titer of the sample to be tested is obtained by using the peak area of the standard curve and the elution curve of the sample to be tested. The affinity chromatography column is packed with protein A, and elution is performed using mobile phase A and mobile phase B, both of which contain arginine.
2. The detection method according to claim 1, characterized in that, The mobile phase A comprises: 45-55 mM Tris-HAc, 140-160 mM NaCl, and 0.45-0.55 M arginine; Preferably, the mobile phase B comprises 110-130 mM HAc and 0.45-0.55 M arginine.
3. The detection method according to claim 2, characterized in that, The mobile phase A comprises: 50 mM Tris-HAc, 150 mM NaCl and 0.5 M arginine.
4. The detection method according to claim 2, characterized in that, The mobile phase B comprises 120 mM HAc and 0.5 M arginine.
5. The detection method according to claim 2, characterized in that, The pH value of the mobile phase A is 6.8-7.
2.
6. The detection method according to claim 2, characterized in that, The pH value of the mobile phase B is 2.9-3.
3.
7. The detection method according to claim 1, characterized in that, The elution process includes: equilibration using mobile phase A during elution of 0-1 min, protein elution using mobile phase B during elution of 1-3 min, and rinsing using mobile phase A during elution of 3-6 min.
8. The detection method according to claim 1, characterized in that, The step of loading the standard onto the affinity chromatography column includes: The standard protein was serially diluted with buffer solution to obtain 7-8 standard proteins of different concentrations, which were then loaded onto the affinity chromatography column for elution and UV detection.
9. The detection method according to claim 8, characterized in that, The concentration range of the standard protein obtained by dilution is 0.1-10 g / L.
10. The detection method according to claim 1, characterized in that, Before loading or eluting, the standard protein, the test sample, mobile phase A, and mobile phase B are filtered using a 0.22 μm filter, respectively.