CN115728433A - Ion exchange chromatography detection method for IgG4 type monoclonal antibody charge heterogeneity - Google Patents
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Abstract
The invention relates to the field of medicines, and provides an ion exchange chromatography detection method for IgG4 monoclonal antibody charge heterogeneity, wherein the detection conditions comprise: and (3) carrying out gradient elution by using a strong cation chromatographic column with sulfonic functional groups and using a mobile phase A and a mobile phase B. The method can effectively detect acidic, neutral and alkaline charge isomers in the IgG4 monoclonal antibody, particularly the Dupilumab monoclonal antibody and biological analogues thereof, and has the advantages of high separation degree, good repeatability, high accuracy, good specificity, simple and quick operation and the like.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and relates to an ion exchange chromatography detection method for the charge heterogeneity of an IgG4 monoclonal antibody.
Background
The therapeutic monoclonal antibody drug is taken as a targeting biological therapeutic drug, and has become a research and development hotspot of pharmaceutical enterprises since the drug is approved to be on the market by FDA in 1986. Dupilumab is the first fully humanized IgG 4-type monoclonal antibody approved by the FDA for the treatment of moderate to severe atopic dermatitis, and inhibits IL-4, IL-13 signaling by binding to the IL-4 Ra subunit. The molecular weight of the monoclonal antibody is about 150kDa, the monoclonal antibody is expressed by adopting eukaryotic cells, various post-translational modification changes can occur in the processes of biosynthesis, fermentation culture, purification, preparation and the like of antibody molecules in cells, including oxidation, deamidation, heavy chain C-terminal lysine residue, isomerization, glycosylation and the like, in addition, the comprehensive factors of size variants such as a breaking body, an aggregation and the like of the antibody molecules cause the difference of charges carried by the antibody molecules, and the characteristic of heterogeneity is finally presented, and the antibody molecules are called as charge isomers. The charge heterogeneity of antibody molecules is closely related to the stability and biological activity thereof, and directly influences the stability of the production process of products, so that the antibody molecules are taken as a key quality attribute (CQA) of the products and are of great interest in the development and production processes of antibody drugs.
Current methods for evaluating charge heterogeneity of monoclonal antibodies include flat isoelectric focusing electrophoresis (IEF), capillary isoelectric focusing electrophoresis (CIEF), ion exchange chromatography (IEX), and the like. Isoelectric focusing electrophoresis (IEF/CIEF) separates charge isomers according to isoelectric point differences of antibody molecules, the detection result of IEF is a gel strip, each charge isomer cannot be accurately quantified and analyzed at present, although CIEF can realize accurate quantification, and the modification causing isoelectric point change, such as incomplete terminal lysine excision, cannot be monitored, such as oxidation modification, and the like, can not be monitored, and in addition, the sample pretreatment of the IEF is complex, and the steps of sample desalting and the like are involved, so that the time is long. The ion exchange chromatography separates charge isomers according to the difference of the surface charges of the antibodies, can comprehensively monitor all modification changes causing heterogeneity, does not need sample pretreatment, and is simple to operate and higher in sensitivity.
Currently, most monoclonal antibodies which are sold on the market belong to IgG1 types, related research analysis technologies are mature, the prior art mainly centers on the analysis and detection of IgG1 type antibodies, generally, the analysis of charge isomers generally utilizes weak cation chromatography mediated by salt ion gradient, a good separation effect can be achieved, and the requirements on process development and product quality evaluation can be met, but the research reports on IgG2 and IgG4 type ion exchange chromatography methods are few, the development of the ion exchange chromatography detection methods by the two types of antibodies has great difficulty, the IgG4 type monoclonal antibodies have high hydrophobicity, and at present, no detection method for the charge heterogeneity of the IgG4 type monoclonal antibodies and the biological analogs thereof with better separation effect and stability exists.
The article by Dadysy et al ("pH gradient ion exchange chromatography high resolution separation of mAb sialylated isomers") discloses that the use of strong cation exchange chromatography based on pH gradients on a ProPac SCX-10 column can be used for high resolution separation of monoclonal antibody sialylated isomers. However, the method for analyzing the charge heterogeneity of the IgG4 monoclonal antibody has the defect that better separation of acidic, neutral and basic isomers cannot be achieved, and the pH gradient separation principle is adopted.
Patent application CN109946410A discloses an ion exchange chromatography detection method for analyzing the charge heterogeneity of monoclonal antibodies by using a weak cation chromatographic column and a Propac WCX-10. However, this method does not allow the separation and detection of the charge isomers of IgG4 monoclonal antibodies.
Therefore, it is necessary to develop a method for detecting the charge heterogeneity of the IgG 4-type monoclonal antibody with better separation effect and stability.
Disclosure of Invention
In view of the above technical situation, the present invention aims to provide a cation exchange chromatography detection method for analyzing the charge heterogeneity of IgG4 monoclonal antibodies, particularly Dupilumab monoclonal antibodies and their biological analogs, which has better resolution, higher specificity, repeatability and accuracy. The detection method comprises the following steps:
the invention provides an ion exchange chromatography detection method for charge heterogeneity of a monoclonal antibody, wherein the monoclonal antibody is an IgG4 type monoclonal antibody, and the detection conditions comprise: adopting a strong cation chromatographic column with sulfonic functional groups to carry out gradient elution by a mobile phase A and a mobile phase B,
wherein the mobile phase A is an ampholyte and a salt buffer solution thereof, preferably MES (2-morpholine ethanesulfonic acid) and a salt buffer solution thereof, phosphoric acid and a salt buffer solution thereof, acetic acid and a salt buffer solution thereof, or a plurality of combinations thereof;
the mobile phase B is an ampholyte and a salt buffer solution thereof, preferably MES (2-morpholine ethanesulfonic acid) and a salt buffer solution thereof, phosphoric acid and a salt buffer solution thereof or acetic acid and a salt buffer solution thereof, or a plurality of combinations thereof;
the salt is a sodium salt or a potassium salt.
In one embodiment of the method of the present invention, the strong cation chromatography column is YMC BioPro IEX SF or Thermo MAbPac SCX-10.
In the method of the present invention, as one of embodiments, the method further comprises: the mobile phase A or the mobile phase B contains acetonitrile, isopropanol, ethanol or methanol with the volume percentage of 0-10%.
In the method of the present invention, as one of embodiments, the method further comprises: the mobile phase A or the mobile phase B contains 5 percent by volume of acetonitrile, isopropanol, ethanol or methanol.
In the method of the present invention, as one of the embodiments, the method further comprises: the mobile phase A or the mobile phase B contains 5% acetonitrile by volume.
In the process of the invention, as one of the embodiments, the mobile phase a is at pH5.7 to 6.2, preferably at pH5.8 to 6.1, optimally at pH6.0.
In the method of the present invention, as one embodiment, the mobile phase a is MES and its salt buffer solution with a concentration of 10-50mM, to which 5% volume of acetonitrile is added, preferably 20-50mM, more preferably 30mM MES and its salt buffer solution with 5% volume of acetonitrile is added; as an embodiment, it is preferred that mobile phase A is 30mM MES, pH6.0,5% by volume acetonitrile.
In the method of the present invention, as one embodiment, the mobile phase B is based on the mobile phase A, and 0.1M to 1M sodium salt or potassium salt solution, preferably 0.5M sodium chloride or potassium chloride solution, is added.
In the method of the present invention, as one embodiment, the mobile phase B is: 30mM MES,0.5M NaCl, pH6.0,5% acetonitrile.
In the method of the present invention, as one of the embodiments, the method further comprises a flow rate of the mobile phase of 0.2 to 1.5mL/min, preferably 0.4 to 0.6mL/min, and further preferably 0.4mL/min.
In the method of the present invention, as one of the embodiments, the method further comprises an elution gradient of:
| time/min | Mobile phase A | Mobile phase B |
| 0min | 93 | 7 |
| 2min | 93 | 7 |
| 57min | 82 | 18 |
| |
40 | 60 |
。
In the method of the present invention, as one of the embodiments, the method further comprises measuring at a column temperature of 20 to 50 ℃, preferably 40 ℃.
In the method of the present invention, as one of the embodiments, the method further comprises using an ultraviolet detector, the detection wavelength is 280nm, and the sample amount is 5 to 100. Mu.g, preferably 25 to 75. Mu.g.
In the method of the present invention, as one of the embodiments, the method further comprises the treatment of the test article: and adding ultrapure water into the IgG4 type monoclonal antibody and the biological analogue thereof, and uniformly mixing to obtain a sample to be detected with the concentration of 2.5 mg/mL.
In the method of the present invention, as one embodiment, the IgG 4-type monoclonal antibody is Dupilumab or a biological analog thereof, or Nivolumab or a biological analog thereof.
In the method, as one embodiment, dupilumab5 μ L is taken, 295 μ L ultrapure water is added, and the mixture is uniformly mixed to obtain a sample to be detected with the concentration of 2.5 mg/mL.
In the method, as one embodiment, nivolumab25 μ L is added into ultrapure water 75 μ L, and the mixture is mixed uniformly to prepare a sample to be tested, wherein the sample to be tested is 2.5 mg/mL.
In the method of the present invention, as one of embodiments, the method further comprises: the high-efficiency detection method for the charge heterogeneity of the Dupirumab or Nivolumab monoclonal antibody and the biological analogue thereof comprises the following detection conditions:
a chromatographic column: YMC BioPro IEX SF or Thermo MAbPac SCX-10
Mobile phase: A-30mM MES, pH6.0,5% acetonitrile;
B-30mM MES,0.5M NaCl, pH6.0,5% acetonitrile
Column temperature: 40 deg.C
Detection wavelength: 280nm
Sample introduction amount: 25 μ g
Flow rate: 0.4mL/min
Gradient of mobile phase:
| time/min | Mobile phase A | Mobile phase B |
| 0min | 93 | 7 |
| 2min | 93 | 7 |
| 57min | 82 | 18 |
| |
40 | 60 |
In the method of the present invention, as one of embodiments, the method comprises:
preparation of a sample to be tested: and adding ultrapure water into Dupilumab or Nivolumab, and uniformly mixing to obtain a sample to be detected with the concentration of 2.5 mg/mL.
Definition of
In the present invention, the term "biosimilar" is also referred to as "biosimilar drug", and the term "biosimilar drug" is defined in the "guidance of biosimilar drug development and pre-evaluation techniques" as a therapeutic biological product having similarity in quality, safety and efficacy to a reference drug approved for registration, and the amino acid sequence of a candidate biosimilar drug should be the same as that of the reference drug in principle. In the present invention, the use of Dupilumab or Nivolumab sequences for drug imitation may be referred to as Dupilumab or Nivolumab biological analogs. The sample to be tested to which the present invention is directed is a solution containing a component having the same sequence or site mutation as that of Dupilumab or Nivolumab antibody, including Dupilumab or Nivolumab and biological analogs thereof.
Compared with the prior art, the invention has the following advantages and effects: the invention provides an ion exchange chromatography detection method aiming at the charge heterogeneity analysis of IgG4 monoclonal antibodies, in particular Dupilumab and biological analogues thereof, which is different from the traditional method which generally utilizes weak cation chromatographic columns to carry out the charge heterogeneity analysis of the monoclonal antibodies, the method adopts strong cation chromatographic columns which take sulfonic groups as functional groups and combines organic reagents which are added into a mobile phase according to a certain proportion to obtain the separation effect superior to a plurality of weak cation chromatographic columns, the method can effectively separate the IgG4 monoclonal antibodies, in particular the acidic, neutral and basic charge isomers of the Dupilumab monoclonal antibodies and the biological analogues thereof, and has the advantages of high separation degree, good repeatability, high accuracy, good specificity, simple and quick operation and the like, and the method has stronger universality and has the separation effect on Nivolumab which is the IgG4 monoclonal antibody superior to the separation of the weak cation chromatographic columns.
Therefore, the detection method successfully realizes the effective detection of the IgG4 monoclonal antibody, particularly the Dupilumab monoclonal antibody and the biological analogue charge isomer thereof, so that the quality attribute of the IgG4 monoclonal antibody, particularly the Dupilumab monoclonal antibody and the biological analogue product thereof can be detected.
Drawings
FIG. 1 is a graph of the acidic, neutral and basic charge isomer profiles of a typical Dupilumab antibody obtained under the assay conditions of example 1;
FIG. 2 is a graph of the acidic, neutral and basic charge isomer profiles of the Dupilumab antibody obtained on Thermo propac Elite WCX under the detection conditions of example 2;
FIG. 3 is a graph of the acidic, neutral and basic charge isomer profiles of the Dupilumab antibody obtained on Thermo propac WCX-10 under the detection conditions of example 2;
FIG. 4 is a graph of the acidic, neutral and basic charge isomer profiles of the Dupilumab antibody obtained on Thermo MAbPac SCX-10 under the detection conditions of example 3;
FIG. 5 is a graph showing the separation effect of acidic, neutral and basic charge isomers of Dupilumab antibody measured at pH5.8, 6.0 and 6.1 in the mobile phase of example 4;
FIG. 6 is a graph showing the separation effect of acidic, neutral and basic charge isomers of Dupilumab antibody measured at concentrations of 20mM and 50mM in mobile phase MES of example 5;
FIG. 7 is a graph showing the effect of separating acidic, neutral and basic charge isomers of Dupilumab antibody measured at flow rates of 0.4 and 0.6mL/min in example 6;
FIG. 8 is a graph showing the separation effect of acidic, neutral and basic charge isomers of Dupilumab antibody measured under the conditions of salt ion gradient of 1.5, 1.0, 0.8 and 0.6mM/min in example 7;
FIG. 9 is a graph showing the effect of separating acidic, neutral and basic charge isomers of Dupilumab antibodies measured at column temperatures of 35, 37, 40, 45 ℃ in example 8;
FIG. 10 is a graph showing the effect of separating acidic, neutral and basic charge isomers of Dupilumab antibodies measured after adding 0%,2% and 5% acetonitrile by volume to the mobile phase of example 9;
FIG. 11 is the acidic, neutral and basic charge isomer profiles of the Dupilumab antibody carboxypeptidase B in example 11 without and after cleavage;
FIG. 12 is a linear regression line graph of the detection method of the present invention in example 12;
FIG. 13 is a graph of the distribution of acidic, neutral and basic charge isomers of Nivolumab mab as measured by the assay conditions of example 14;
FIG. 14 is a graph of the acid, neutral and basic charge isomer distribution of Nivolumab mab detected on a weak cation chromatography column from Thermofisiher, inc. in example 14.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Instruments and reagents
TABLE 1
Example 1
This example is an experiment for detecting charge isomers of Dupilumab antibody using a YMC BioPro IEX SF column, and the procedure is as follows
(1) Preparation of sample 1 to be tested (also referred to as "treatment of sample 1"): and adding 295 mu L of ultrapure water into 5 mu L of Dupilumab monoclonal antibody (batch No. 9L 003K), and uniformly mixing to obtain a sample to be detected with the concentration of 2.5 mg/mL.
(2) Determining chromatographic conditions: the detection chromatographic conditions are shown in table 2.
TABLE 2
The results are shown in FIG. 1, and the acidic, neutral and basic charge isomers of Dupilumab can be effectively separated by using the above detection conditions.
Example 2
This example compares example 1 and tests the separation effect of Dupilumab on a weak cation chromatography column under the same mobile phase conditions.
(1) Treatment of the test article: the same as in example 1.
(2) The detection chromatographic conditions are shown in Table 3
TABLE 3
As shown in FIG. 2, the Dupilumab antibody showed only a small separation of acid peaks and no separation of base peaks on Thermo propac Elite WCX.
In addition, the same mobile phase and salt ion change gradient were used to separate Dupilumab antibody on Thermo propac WCX-10 weak cation chromatographic column, and the results are shown in fig. 3, which shows that the chromatographic column is used for evaluating the charge heterogeneity of IgG1 type antibody, but has no separation effect on acidic, neutral and basic charge isomers of Dupilumab antibody.
Example 3
This example investigates the separation effect of different strong cation chromatography columns on Dupilumab charged isomers.
The sample was treated as in example 1, and Thermo MAbPac SCX-10 was selected as the column, except that the column was different, and the other detection conditions were the same as in example 1.
The results are shown in FIG. 4, and the separation effect of Thermo MAbPac SCX-10 strong cation chromatographic column on acidic, neutral and basic charge isomers of Dupilumab is equivalent to that of example 1 under the same detection conditions.
Example 4
This example investigates the effect of different mobile phase pH values on separation of Dupilumab charge isomers.
The sample was treated as in example 1, and the chromatographic conditions are shown in Table 4:
TABLE 4
The detection results are shown in fig. 5, the mobile phase has pH5.8, 6.0 and 6.1, the detection conditions have similar separation effects on the acidic, neutral and basic isomers of the Dupilumab antibody, and can meet the separation requirements, and pH6.0 is the optimal pH in view of the shape of each peak.
Example 5
This example investigates the effect of different MES concentrations on the separation of Dupilumab charge isomers.
The samples were treated as in example 1, the only difference between the chromatographic conditions and example 1 was the MES concentration, and 20 and 50mM MES detection conditions were examined.
As shown in FIG. 6, the separation effect of the acid, main and base peaks of the Dupilumab antibody was comparable to that of the MES concentration of 30mM (see FIG. 1).
Example 6
This example investigates the effect of different flow rates on the separation of Dupilumab charge isomers.
The sample is treated as in example 1, and the only difference between the chromatographic conditions and example 1 is the flow rate, and since the flow rate of the chromatographic column is preferably 0.2-0.8mL/min, the present invention considers the detection conditions of 0.4 and 0.6 mL/min.
As shown in FIG. 7, when the flow rate is increased from 0.4mL/min to 0.6mL/min, the separation effect of the acid-base peak close to the main peak is slightly decreased, but the total separation effect is not changed, and the separation effect can also be achieved.
Example 7
This example investigates the effect of varying gradients of different salt ions on the separation of Dupilumab charge isomers. The salt ion gradient refers to the ratio of the difference in salt concentration of mobile phase B rising from 7% to 18% to the time used, and the salt ion gradient of example 1 is 1.0mM/min.
The samples were treated as in example 1. The chromatographic conditions differed from those of example 1 in that the gradient of change in salt ion of mobile phase B was varied, and the gradient of change was examined at 0.6, 0.8, 1.0 and 1.5 mM/min.
As shown in FIG. 8, the degree of separation of the acid-base peak decreased with the increase of the salt ion gradient from 0.6 to 1.5mM/min, but both the separation effect and the optimum salt ion gradient of 1.0mM/min were achieved for both analysis time and peak resolution.
Example 8
This example examined the effect of different column temperatures on the separation of Dupilumab charge isomers.
The sample was treated as in example 1, and the chromatographic conditions were different from those in example 1 in the column temperature, and 35 ℃,37 ℃,40 ℃ and 45 ℃ were examined.
As shown in fig. 9, the column temperature is increased from 35 ℃ to 40 ℃, the peak separation effect of the base peak is slightly increased, and when the column temperature is increased to 45 ℃, the separation effect is substantially the same as that at 35 ℃, the separation effect can be achieved, the improvement of the column temperature on the separation effect of the acid-base peak is limited, and the column temperature at 40 ℃ is the optimal detection temperature.
Example 9
This example is an experiment for testing the separation effect of Dupilumab charged isomers at different acetonitrile concentrations.
The only difference from example 1 is that the acetonitrile addition ratio of the mobile phase a and the mobile phase B is that the influence of the concentrations (volume percentages) of 0%,2% and 5% acetonitrile on the separation results is examined, the detection spectrum is shown in fig. 10, the analysis results are shown in table 5, the experimental requirements and the detection of the process samples can be met even under the condition of not adding acetonitrile, but the separation degree of the detection results can be obviously improved after 5% acetonitrile is added.
TABLE 5
| Acetonitrile concentration (volume percentage) | Separation degree of acid peak A1 and main peak M | Peak to valley ratio of |
| 0％ | 0.58 | 1.25 |
| 2％ | 0.62 | 1.29 |
| 5％ | 0.89 | 1.45 |
Wherein the peak-to-valley ratio of the acid peak A1 = peak height of the A1 peak/valley height adjacent to the main peak.
Example 10
This example is to verify the repeatability of the detection method.
The sample was treated in the same manner as in example 1, with mobile phases having 30mM MES concentration and 20mM MES concentration being set as the chromatographic detection conditions, and the other detection conditions being unchanged. 8 test samples are prepared, and compared with the retention time of a main peak and the content change of acidic, neutral and basic charge isomers after detection, the results are shown in tables 6 and 7, the repeatability of the detection method of the 30mM MES mobile phase and the 20mM MES mobile phase is reflected, and the repeatability of the detection method is better.
TABLE 6.20mM MES +5% acetonitrile as mobile phase
| Sample(s) | Main peak retention time/min | Acid peak content% | Content of main peak% | Content of alkali |
| 1 | 32.186 | 31.062 | 61.105 | 7.833 |
| 2 | 32.238 | 30.309 | 61.512 | 8.179 |
| 3 | 32.222 | 31.193 | 61.173 | 7.634 |
| 4 | 31.955 | 31.199 | 61.142 | 7.659 |
| 5 | 31.984 | 30.969 | 61.403 | 7.728 |
| 6 | 32.039 | 31.221 | 61.067 | 7.712 |
| 7 | 31.968 | 31.326 | 61.065 | 7.609 |
| 8 | 31.947 | 31.296 | 61.978 | 7.726 |
| RSD value | 0.39％ | 1.08％ | 0.30％ | 2.21％ |
TABLE 7.30mM MES+5% acetonitrile as mobile phase
Example 11
The embodiment is to verify the specificity of the detection method. During the expression process of the monoclonal antibody, most of the lysine at the C-terminal of the heavy chain of the monoclonal antibody is cut by the intracellular carboxypeptidase, but a small amount of lysine remains, so that the basic charge isomer of the antibody is increased, namely, the lysine residue at the C-terminal of the heavy chain of the antibody is one of the reasons for the basic charge isomer. Specificity was therefore verified by sample testing after treatment of Dupilumab with carboxypeptidase B.
Treatment of sample 2: dupilumab 5. Mu.L (lot 9L 003K) was taken, and 295. Mu.L of pH7.0 and 25mM Tris solution was added thereto and mixed well to obtain 2.5mg/mL Dupilumab sample.
Treatment of Dupilumab carboxypeptidase B digested sample: and taking 150 mu L of the test solution, adding 4 mu L of carboxypeptidase B solution, and mixing uniformly.
The Dupilumab sample 2 and the enzyme-digested sample were incubated at 37 ℃ for 60min in a blank solution of 25mM Tris, pH 7.0.
The detection chromatographic conditions are as shown in table 1, the detection results are shown in fig. 11, compared with the results of enzyme digestion by carboxypeptidase B, after the test sample 2 is subjected to enzyme digestion, the corresponding alkaline peak is lowered, and the peak is shown by an arrow, which is in line with theoretical expectation, and therefore, the detection method can effectively separate a small amount of alkaline charge isomers caused by heavy chain C-terminal lysine residue, and compared with the chromatogram of a blank solution, no interference peak is generated at the peak position of the test sample, which indicates that the method can be specially used for detecting the Dupilumab antibody.
Example 12
This embodiment verifies the linearity of the detection method.
The test article was treated and tested under the same conditions as in example 1. Inspecting the linearity of the sample in the range of 25-75 μ g, preparing 3 parts of sample solution, measuring each sample once according to the sample volume of 10 μ L,15 μ L,20 μ L,25 μ L,30 μ L, calculating the average peak area of acidic, neutral and basic isomers with different sample amount, the unit is mAU · s, and taking the sample amount of the sample as abscissa, the average peak area is calculatedThe area is the ordinate, linear regression is respectively carried out on the acidic charge variant, the neutral charge variant and the alkaline charge isomer, and a linear correlation coefficient R is obtained according to a regression curve 2 The statistical results of the specific peak areas and correlation coefficients are shown in Table 8, and the regression curves are shown in FIG. 12, which illustrate the linear regression coefficient R of each charge variant with the sampling amount in the range of 25-75 μ g using this detection method 2 Are all at>Within an acceptable range of 0.99.
TABLE 8
| Sample volume | Area of acidic peak | Area of neutral peak | Area of basic peak |
| 25μg | 1419.014 | 2838.076 | 344.150 |
| 37.5μg | 2132.289 | 4266.835 | 496.096 |
| 50μg | 2816.487 | 5690.367 | 648.115 |
| 62.5μg | 3464.802 | 7130.874 | 803.470 |
| 75μg | 4140.146 | 8520.819 | 939.448 |
| R 2 | 0.9997 | 1 | 0.9995 |
Example 13
The present embodiment verifies the accuracy of the detection method.
The test article was treated and tested under the same conditions as in example 1. Respectively preparing 3 parts of sample solutions, measuring each part of sample solution once according to the sample injection volume of 12 mu L and 18 mu L, wherein the corresponding sample injection amount is respectively 30 mu g and 45 mu g, substituting the peak areas into a regression equation obtained by linear verification, respectively calculating the peak areas of acidic, neutral and basic charge isomers, and calculating the corresponding sample injection amount, and dividing the actual sample injection amount by the theoretical sample injection amount to obtain the recovery rate (%), wherein the statistical result is shown in table 9, and the recovery rates of all the components under the conditions of the sample injection amounts are within an acceptable range of 90-110%.
TABLE 9
Example 14
This example analyzes the charge heterogeneity of Nivolumab using the same detection conditions, and tests the versatility of the method.
Treatment of test article 3: nivolumab monoclonal antibody (batch number ABR 1014) 25. Mu.L is taken, added into 75. Mu.L of ultrapure water and mixed evenly to prepare a sample 3 solution with the concentration of 2.5 mg/mL.
The separation according to the detection conditions of example 1 is carried out, the obtained results are shown in fig. 13, and it can be seen from the figure that the acidic, neutral and basic charge isomers of Nivolumab of IgG4 type can be effectively separated by using the same detection conditions, and compared with the detection results of Nivolumab monoclonal antibody mentioned in the application information of the weak cation chromatographic column (Propac Elite WCX) of thermoliser company, as shown in fig. 14, the method of the present invention has better separation effect on Nivolumab antibody, which indicates that the method of the present invention has universality in the charge heterogeneity analysis of IgG4 type antibody.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (17)
1. An ion exchange chromatography detection method for IgG4 monoclonal antibody charge heterogeneity, characterized in that the detection conditions comprise: carrying out gradient elution by using a strong cation chromatographic column with sulfonic functional groups and a mobile phase A and a mobile phase B;
the mobile phase A is an ampholyte and a salt buffer solution thereof; preferably MES (2-morpholinoethanesulfonic acid) and a salt buffer solution thereof, phosphoric acid and a salt buffer solution thereof, or acetic acid and a salt buffer solution thereof, or a combination of a plurality of these;
the mobile phase B is an ampholyte and a salt buffer solution thereof; preferably MES (2-morpholine ethanesulfonic acid) and a salt buffer solution thereof, phosphoric acid and a salt buffer solution thereof, or acetic acid and a salt buffer solution thereof, or a combination of a plurality of the same;
the salt is a sodium salt or a potassium salt.
2. The method of claim 1, wherein the strong cation chromatography column is YMC BioPro IEX SF or Thermo MAbPac SCX-10.
3. The method of claim 1, further comprising: the mobile phase A or the mobile phase B contains acetonitrile, isopropanol, ethanol or methanol with the volume percentage of 0-10%.
4. The method of claim 3, further comprising: the mobile phase A or the mobile phase B contains 5 percent by volume of acetonitrile, isopropanol, ethanol or methanol.
5. The method of claim 4, further comprising: the mobile phase a or the mobile phase B contained 5% by volume of acetonitrile.
6. The method according to claim 1, wherein the mobile phase a has a ph of 5.7-6.2, preferably 5.8-6.1, most preferably ph6.0.
7. The method according to claim 6, wherein the mobile phase A is MES with a concentration of 10-50mM and its salt buffer solution added with 5% acetonitrile by volume; MES and its salt buffer solution of preferably 20-50mM, more preferably 30mM is added with 5% acetonitrile by volume; the optimum mobile phase A was 30mM MES, pH6.0,5% by volume acetonitrile.
8. The method according to claim 1, wherein the mobile phase B is based on the mobile phase A, and a 0.1M to 1M solution of a sodium or potassium salt is added, preferably a 0.5M solution of sodium or potassium chloride.
9. The method according to claim 8, wherein the mobile phase B is: 30mMMES,0.5M NaCl, pH6.0,5% by volume acetonitrile.
10. The method according to claim 1, wherein the method further comprises a mobile phase flow rate of 0.2-1.5mL/min, preferably 0.4-0.6mL/min, further preferably 0.4mL/min.
11. The method of claim 1, further comprising an elution gradient of:
。
12. The method according to claim 1, further comprising measuring at a column temperature of 20-50 ℃, preferably 40 ℃.
13. The method of claim 1, further comprising using an ultraviolet detector with a detection wavelength of 280nm; the amount of sample is 5-100. Mu.g, preferably 25-75. Mu.g.
14. The method of claim 1, further comprising processing the test article: and adding ultrapure water into the IgG4 type monoclonal antibody and the biological analogue thereof, and uniformly mixing to obtain a sample to be detected with the concentration of 2.5 mg/mL.
15. The method of claim 1, wherein the IgG 4-type monoclonal antibody and biological analog thereof is Dupirumab and a biological analog thereof, or Nivolumab and a biological analog thereof.
16. The method of claims 1-15, further comprising the following conditions for efficient detection of charge heterogeneity of Dupilumab or Nivolumab and its biological analogs:
a chromatographic column: YMC BioPro IEX SF or Thermo MAbPac SCX-10
Mobile phase: a:30mM MES, pH6.0,5% acetonitrile;
b:30mM MES,0.5M NaCl, pH6.0,5% acetonitrile
Column temperature: 40 deg.C
Detection wavelength: 280nm
Sample introduction amount: 25 μ g
Flow rate: 0.4mL/min
Gradient of mobile phase:
。
17. The method of claim 16, wherein the method comprises:
preparation of a sample to be tested: and adding ultrapure water into Dupilumab or Nivolumab, and uniformly mixing to obtain a sample to be detected with the concentration of 2.5 mg/mL.
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