CN114544839A - Charge variant detection method for anti-human nerve growth factor antibody - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
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Abstract
The invention discloses a charge variant detection method of an anti-human nerve growth factor antibody. The conditions are as follows: thermo DIONEX Ultimate 3000 high performance liquid chromatograph; sepax Protemix SCX-NP5 chromatography column; the column temperature is 25 ℃; mobile phase a was a 4mM Tris-4mM imidazole-4 mM piperazine solution (pH 5.0); the mobile phase B is a 4mM Tris-4mM imidazole-4 mM piperazine-0.25M NaCl solution; elution procedure: 100% by volume of mobile phase a from 0 min; 9 min-15.5 min, 80% mobile phase A and 20% mobile phase B. The method can be used for rapidly detecting a large batch of samples, and has the advantages of high accuracy, good repeatability, stability after sample treatment, simple operation, low cost, high universality and good applicability.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a charge variant detection method of an anti-human nerve growth factor antibody.
Background
Anti-human nerve growth factor antibodyIs IgG4The monoclonal antibody blocks a pain transmission pathway by binding with nerve growth factor, and has a wide therapeutic effect on pain diseases such as osteoarthritis, fracture pain, rheumatoid arthritis, gout joint pain, neuropathic pain and the like. During production, storage, transportation, etc., the monoclonal antibody may interact with the material in solution and the surface residues may undergo various chemical or enzymatic modifications, resulting in the monoclonal antibody becoming a mixture of charge variants with slight electrostatic differences. Charge variants are generally classified as acidic or basic variants based on their isoelectric Point (PI) relative to the main peak. In general, acidic variants have a lower PI relative to the main peak, while basic variants have a higher PI relative to the main peak. The modification type of the surface residue is determined by analyzing different components of the modified surface residue.
Currently, ion exchange chromatography is widely used in the biopharmaceutical industry as the "gold standard" for monoclonal antibody charge variant analysis. The method adopts a high performance liquid chromatograph, and the dissociative substances are separated and measured through ion exchange. Based on the difference of mobile phase, two types of salt elution and pH elution are divided. Based on the difference of elution program, two types of gradient elution and isocratic elution are divided.
According to the conventional method in the field, an IgG antibody with the PI of 7.5-8.0 is analyzed by adopting a cation chromatographic column; conversely, for IgG antibodies with PI lower than 7.5, an anion chromatographic column was used.
Disclosure of Invention
The invention aims to provide a charge variant detection method of an anti-human nerve growth factor antibody.
In order to achieve the above object, the present invention provides a method for detecting a charge variant of an anti-human nerve growth factor antibody, which is characterized in that a detection sample containing the anti-human nerve growth factor antibody is subjected to HPLC detection;
wherein the chromatograph used in HPLC detection is a Thermo DIONEX Ultimate 3000 high performance liquid chromatograph; the chromatographic column is Sepax Protemix SCX-NP5 with specification of 4.6mm multiplied by 250mm multiplied by 5 mu m; the temperature of the chromatographic column is 25 ℃;
mobile phase: the mobile phase A is a 4mM Tris-4mM imidazole-4 mM piperazine solution, and the pH value of the mobile phase A is 5.0; the mobile phase B is a 4mM Tris-4mM imidazole-4 mM piperazine-0.25M NaCl solution;
flow rate of mobile phase: 1.00 mL/min;
UV detection wavelength: 280 nm;
elution procedure: starting from 0min, the mobile phase is 100% mobile phase A by volume; 9 min-15.5 min, wherein the mobile phase comprises 80% of mobile phase A and 20% of mobile phase B; 16.5 min-24.5 min, and the mobile phase is 100% mobile phase B.
Furthermore, the concentration of the anti-human nerve growth factor antibody in the detection sample containing the anti-human nerve growth factor antibody is 0.750 mg/ml-2.250 mg/ml.
The anti-human nerve growth factor antibody has a PI of about 6.5, and the inventor selects an anion chromatographic column to analyze the charge variant thereof according to common knowledge. However, the chromatogram shows that the inflection point between the main peak and the acid peak is not obvious, the alkali peak is only one, the resolution is low, and the result is not ideal. Therefore, the inventors of the present invention tried to exchange the anion exchange chromatography column for the cation exchange chromatography column for the charge variant analysis of the anti-human nerve growth factor antibody, and the results showed that the inflection point between the components was more obvious than that of the anion column. And then continuously optimizing the mobile phase and the elution method to obtain the technical scheme of the invention.
The detection method provided by the invention performs charge variant analysis on the antibody with the isoelectric point less than 7 by using the strong cation exchange chromatographic column, and the result of the analysis is that more charge variant components can be separated compared with the anion exchange chromatographic column, the inflection points between peaks are clearer and clearer, the stable and automatic integration is facilitated, the analysis of the charge variants of the human nerve growth factor antibody is facilitated, and the control of the product quality is facilitated.
The detection method provided by the invention is characterized in that NaCl is added into a mobile phase 1(4mM Tris-4mM imidazole-4 mM piperazine solution) (namely, a mobile phase B is formed, and the concentration of the NaCl in the mobile phase A is equal to that in the mobile phase A: a4 mM Tris-4mM imidazole-4 mM piperazine solution (pH5.0) formed a binary mobile phase. Compared with a ternary mobile phase formed by the mobile phase A, the mobile phase 1 and a NaCl solution, the elution time is shorter, the inflection point is more obvious during elution, the elution degree is higher, and the elution effect is better.
The detection method provided by the invention combines salt elution and pH elution, gradient elution and isocratic elution, the proportion of the mobile phase B is uniformly increased within 0-9 min, the volume proportion (80: 20) of the mobile phase A and the mobile phase B is fixed within 9-15.5 min, namely the pH and the salt ion concentration are kept constant after certain conditions are reached, and the charge variants of the anti-human nerve growth factor antibody are sequentially eluted from the strong cation chromatographic column. Compared with the method only depending on salt elution or pH elution, gradient elution or isocratic elution, the signal value of the alkaline variant is lower, and the separation degree between the components is better.
The detection method provided by the invention can be used for rapidly detecting mass samples, has the advantages of high detection speed, high accuracy, good repeatability, stability after sample treatment, simple operation, low cost, high universality and good applicability, and has great application value in quality control of drug research and development and production. The required instruments, reagents and the like are all conventional configurations of medical enterprises and research institutions, and are suitable for large-scale popularization and use.
The anti-human nerve growth factor antibody has good linear relation with different charge variant components within the range of 0.75mg/ml to 2.25mg/ml, and can meet the detection requirements of anti-human nerve growth factor antibody charge variants with different concentration levels. Example 1 verifies that the method of the invention has high stability, good repeatability and high accuracy.
Drawings
FIG. 1 is a HPLC detection profile of anti-human nerve growth factor antibody. 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), 2 is followed by a Base Peak (BP).
FIG. 2 is a HPLC detection profile of mobile phase A.
FIG. 3 is a HPLC detection profile of the sample matrix.
FIG. 4 is a HPLC detection profile of a sample solution at a concentration of 0.750 mg/ml. 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), 2 is followed by a Base Peak (BP).
FIG. 5 is a HPLC detection profile of a sample solution at a concentration of 1.125 mg/ml. 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), and 2 is followed by a Base Peak (BP).
FIG. 6 is a HPLC detection profile of a sample solution at a concentration of 1.500 mg/ml. 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), 2 is followed by a Base Peak (BP).
FIG. 7 is a HPLC chromatogram of a sample solution at a concentration of 1.875 mg/ml. 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), 2 is followed by a Base Peak (BP).
FIG. 8 is a HPLC detection profile of a sample solution at a concentration of 2.250 mg/ml. 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), 2 is followed by a Base Peak (BP).
FIG. 9 is a graph of acid peak regression. The x-axis is the sample concentration (mg/ml) and the y-axis is the acid peak component area (mAU. min).
FIG. 10 is a graph of the main peak regression. The x-axis is the sample concentration (mg/ml) and the y-axis is the major peak component area (mAU. min.).
FIG. 11 is a graph of the base peak regression. The x-axis is the sample concentration (mg/ml) and the y-axis is the base peak component area (mAU. min).
FIG. 12 is a HPLC detection profile of a sample to be tested (i.e., anti-human nerve growth factor antibody injection). 2 is the Main Peak (MP), 2 is preceded by an Acid Peak (AP), 2 is followed by a Base Peak (BP).
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1:
1 preparation of solution
Mobile phase A: namely a 4mM Tris-4mM imidazole-4 mM piperazine solution (pH5.0), taking 0.63g of Tris-HCl, 0.27g of imidazole and 0.34g of piperazine, adding ultrapure water for dissolving, adjusting the pH to 5.0 by hydrochloric acid, and adding water to a constant volume of 1000 ml;
mobile phase B: namely, 4mM Tris-4mM imidazole-4 mM piperazine-0.25M NaCl solution, 0.63g Tris-HCl, 0.27g imidazole, 0.34g piperazine and 14.61g NaCl are taken, and water is added to the solution to be constant volume of 1000 ml.
2 sample and sample dilution
Anti-human nerve growth factor antibody (CN 109593133B A method for separating charge isomers of anti-human nerve growth factor antibody) the constructed vector is transfected into CHO cells and purified by the methods of example 1 and example 3 to obtain the anti-human nerve growth factor antibody) is diluted to the concentration of 1.50mg/mL by using mobile phase A.
3HPLC detection
The detection conditions are as follows:
chromatograph: thermo DIONEX Ultimate 3000 high performance liquid chromatograph (including online degasser, quaternary pump, autosampler, column oven, DAD detector and Chromeleon 7 workstation);
a chromatographic column: sepax Protemix SCX-NP5 with specification of 4.6mm × 250mm × 5 μm, i.e. inner diameter 4.6mm, column length 250mm, and filler particle size 5 μm;
temperature of the column: 25 ℃;
mobile phase: the same as above;
elution procedure: the elution volume ratios are shown in table 1;
TABLE 1 elution volume ratio Table for mobile phase A and mobile phase B
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 2 | 100 | 0 |
| 9 | 80 | 20 |
| 15.5 | 80 | 20 |
| 16.5 | 0 | 100 |
| 20.5 | 0 | 100 |
| 20.6 | 100 | 0 |
| 24.5 | 100 | 0 |
Flow rate of mobile phase: 1.00 mL/min;
sample introduction amount: 50 mu L of the solution;
UV detection wavelength: 280 nm.
4 specificity test
The sample matrix (sample matrix is an aqueous solution containing 8% sucrose, 0.05% Tween 20, and 10mM sodium acetate (pH 5.0)) diluted by the same times in the diluted sample, mobile phase A, and reference sample were each measured under the above-described measurement conditions. The detection map is shown in figure 1, figure 2 and figure 3. In FIG. 1, 2 is the Main Peak (MP), 2 is preceded by the Acid Peak (AP), and 2 is followed by the Base Peak (BP).
By comparing fig. 1, 2 and 3, it can be seen that: the mobile phase A and the sample matrix have no absorption peak at the acid peak, the main peak and the alkali peak, and the sample has obvious absorption peaks at corresponding positions, which indicates that the specificity of the method meets the requirements. Meanwhile, fig. 1 shows that an obvious inflection point exists between a main peak and an acid peak, a plurality of small peaks are arranged in an alkali peak, the retention time of the main peak is 11.753min, the retention time of the acid peak is 11.447min, the retention time of the alkali peak 1 is 12.487min, and the separation degrees of the main peak and the acid peak, and the separation degrees of the main peak and the alkali peak 1 are respectively 0.45 and 2.34, so that the method solves the problems existing in the anion exchange chromatographic column.
5 systematic applicability experiment
1 part of the diluted sample was taken, measured 5 times under the above conditions, and the retention time, area ratio and area of each component and the respective relative standard deviation RSD were calculated, and the results are shown in Table 2.
TABLE 2 table of system applicability experiment results
Note: wherein AP is an acid peak, MP is a main peak, BP1 is an alkali peak 1, BP2 is an alkali peak 2, BP3 is an alkali peak 3, BP4 is an alkali peak 4, BP5 is an alkali peak 5, and BP is an alkali peak. BP includes BP1, BP2, BP3, BP4 and BP 5.
As can be seen from Table 2, the retention time, the area ratio and the area RSD of each component are all less than 2.0%, which shows that the system of the method has good applicability.
6 repeatability test
6 parts of the diluted sample were taken and measured under the above-mentioned conditions, and the area ratio of each component and its RSD were calculated, and the results are shown in Table 3.
TABLE 3 repeatability test results Table
Note: wherein AP is an acid peak, MP is a main peak, and BP is a base peak.
As can be seen from Table 3, the area ratio RSD of each component is less than 4.0%, which shows that the method has good repeatability.
7 intermediate precision experiment
The 3 experimenters respectively take 6 parts of the mobile phase A for the anti-human nerve growth factor antibody to prepare a solution with the concentration of 1.5mg/ml, respectively measure according to the detection conditions, calculate the area ratio of each component and the RSD thereof, and the result is shown in Table 4.
TABLE 4 intermediate precision experimental results table
Note: a1, A2 and A3 are the codes of 3 experimenters. AP is the acid peak, MP is the main peak, and BP is the base peak.
As can be seen from Table 4, the area ratio RSD of each component obtained by the experiments of 3 experimenters is less than 4.0 percent, which shows that the intermediate precision of the method is good.
8 Linear experiment
The anti-human nerve growth factor antibody is prepared into 6 parts of solutions with the concentrations of 0.750, 1.125, 1.500, 1.875 and 2.250mg/ml by using the mobile phase A, the solutions are respectively measured according to the detection conditions, a regression curve is drawn by using the concentration of x and the component area of y, and the result is shown in Table 5.
TABLE 5 results of the Linear experiment
Note: wherein AP is an acid peak, MP is a main peak, and BP is a base peak.
The HPLC detection chromatograms of the sample solutions with different concentration gradients are shown in fig. 4-8, and the regression curves of different components are shown in fig. 9-11. As can be seen from FIGS. 4-11 and Table 5, the area RSD of each component in the range of 0.750 mg/ml-2.250 mg/ml is less than 4.0%, and the components are in good linear relation, and the regression equation of the acid peak is that y is 10.2695x +1.3200, R is20.9999; the regression equation of the main peak is that y is 14.9911x +0.9571, R20.9998; the regression equation of the base peak is that y is 12.2729x-1.6871, R2＝0.9992。
9 accuracy test
The anti-human nerve growth factor antibody is prepared into 6 parts of solutions with the concentrations of 0.750, 1.125, 1.500, 1.875 and 2.250mg/ml by using the mobile phase A, the detection is carried out according to the detection conditions, the area recovery rate and the corresponding area recovery rate RSD of each component at each concentration level are calculated, and the results are shown in Table 6.
TABLE 6 accuracy test results table
Note: wherein AP is an acid peak, MP is a main peak, and BP is a base peak.
As can be seen from Table 6, the recovery rates RSD of the components at different concentration levels are less than 4.0%, which indicates that the method has high accuracy.
10 stability test
The diluted samples were taken and treated according to the above conditions, and when they were stored at room temperature for 6 hours, 12 hours, and 24 hours, respectively, the measurement was carried out according to the above detection conditions, and the relative deviation RD of the area ratio of each component was calculated, and the results are shown in table 7.
TABLE 7 stability test results Table
Note: wherein AP is an acid peak, MP is a main peak, and BP is a base peak.
It can be seen that the RD of each component in the sample is less than 4.0% at different times, which indicates that the stability of the sample is good.
11 Charge variant detection of test samples
The sample to be tested (injection prepared from anti-human nerve growth factor antibody by conventional method) was diluted to 1.5mg/ml with mobile phase A, and the area ratio of each component was calculated according to the above test conditions, and the results are shown in FIG. 12. Comparing fig. 1 and fig. 12, it can be seen that the HPLC profile of the sample to be detected is consistent with the HPLC profile of fig. 1, which indicates that the method can be used for detecting the charge heterovariant of the anti-human nerve growth factor antibody, and is of great significance for quality control thereof.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (2)
1. A charge variant detection method of anti-human nerve growth factor antibody is characterized in that a detection sample containing the anti-human nerve growth factor antibody is subjected to HPLC detection;
wherein the chromatograph used in HPLC detection is a Thermo DIONEX Ultimate 3000 high performance liquid chromatograph; the chromatographic column is Sepax Protemix SCX-NP5 with specification of 4.6mm multiplied by 250mm multiplied by 5 mu m; the temperature of the chromatographic column is 25 ℃;
mobile phase: the mobile phase A is a 4mM Tris-4mM imidazole-4 mM piperazine solution, and the pH value of the mobile phase A is 5.0; the mobile phase B is a 4mM Tris-4mM imidazole-4 mM piperazine-0.25M NaCl solution;
flow rate of mobile phase: 1.00 mL/min;
UV detection wavelength: 280 nm;
elution procedure: starting from 0min, the mobile phase is 100% mobile phase A by volume; 9min to 15.5min, the mobile phase is 80 percent of mobile phase A and 20 percent of mobile phase B, 16.5min to 24.5min, and the mobile phase is 100 percent of mobile phase B.
2. The method for detecting a charge variant of an anti-human nerve growth factor antibody according to claim 1, wherein the concentration of the anti-human nerve growth factor antibody in the test sample containing the anti-human nerve growth factor antibody is 0.750mg/ml to 2.250 mg/ml.
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| US20150110799A1 (en) * | 2013-10-18 | 2015-04-23 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same |
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