CN114563489B - Method for detecting recombinant protein charge variant and application thereof - Google Patents
Method for detecting recombinant protein charge variant and application thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title description 4
- 102100032530 Glypican-3 Human genes 0.000 claims abstract description 51
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 claims abstract description 51
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims abstract description 43
- 239000012535 impurity Substances 0.000 claims abstract description 34
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 29
- 230000027455 binding Effects 0.000 claims abstract description 24
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 238000003908 quality control method Methods 0.000 claims abstract description 5
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 3
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 238000010828 elution Methods 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 17
- 230000000694 effects Effects 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 12
- 238000001228 spectrum Methods 0.000 claims description 11
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 5
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000005342 ion exchange Methods 0.000 claims 1
- 239000000710 homodimer Substances 0.000 abstract description 15
- 238000000926 separation method Methods 0.000 abstract description 15
- 239000003814 drug Substances 0.000 abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 238000004925 denaturation Methods 0.000 abstract 1
- 230000036425 denaturation Effects 0.000 abstract 1
- 235000021317 phosphate Nutrition 0.000 abstract 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 24
- 229940125644 antibody drug Drugs 0.000 description 9
- 239000012071 phase Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 7
- 230000009870 specific binding Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000000533 capillary isoelectric focusing Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 239000012526 feed medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000012799 strong cation exchange Methods 0.000 description 1
- 238000012437 strong cation exchange chromatography Methods 0.000 description 1
- 238000002305 strong-anion-exchange chromatography Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/26—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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/96—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 using ion-exchange
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
Abstract
The invention provides a method for detecting charge variants capable of specifically binding GPC3 and CD3 proteins and application thereof. Analyzing a protein to be detected by a high performance liquid-ion exchange chromatography to obtain a chromatogram, and determining the content of homodimer impurities and charge variants of the protein to be detected; mobile phases used in high performance liquid-ion exchange chromatography include mixed solutions of phosphates; mobile phase a and mobile phase B are constituted by different mixing ratios. The invention can be suitable for analyzing the charge variant capable of specifically combining GPC3 and CD3 proteins with complex structures, solves the problems that sample denaturation is caused by reagent or pretreatment, separation of charge isomers cannot be realized by using the traditional high-salt elution-ion exchange chromatography, and the like, can be used in the field of quality control of medicaments, ensures the batch consistency of medicament production, monitors the safety and effectiveness of clinical medicaments, and is applied to detecting impurities and charge variants capable of specifically combining GPC3 and CD3 protein homodimers.
Description
Technical Field
The invention belongs to the technical field of recombinant protein detection, and relates to a method for detecting impurities and charge variants capable of specifically combining GPC3 and CD3 protein homodimers and application thereof.
Background
Recombinant bispecific antibody drugs expressed by eukaryotic systems consist of two distinct heavy chains that are randomly combined to contain homo-dimer impurities and charge-heterogeneous target products. The homodimer impurity is a key quality attribute of the recombinant bispecific antibody drug, and has important influence on stability, solubility, immunogenicity, in-vivo and in-vitro biological activity, pharmacokinetic function exertion and the like of the recombinant bispecific antibody drug. The analysis of homodimers of recombinant bispecific antibody drugs can understand the impurity conditions in the drugs, and is one of the indispensable projects for the quality control of recombinant bispecific antibody drugs.
The current detection methods for bispecific antibody drug charge heterogeneity and homodimer impurity are mainly three, namely plate gum isoelectric focusing electrophoresis, ion exchange chromatography, capillary isoelectric focusing electrophoresis (drug biotechnology, 2018, 25 (5): 461-466). The isoelectric focusing electrophoresis method of the plate gel can rapidly and intuitively display isoelectric point zone, has wider popularization, but has complex operation, low resolution and difficult accurate quantification. The capillary isoelectric focusing electrophoresis method has improved resolution and accurate quantification, but the band is widened and deformed due to the fact that after the focusing process, each component separated by focusing needs to be pushed to a detection window by mechanical or electric field force for detection. Ion exchange chromatography can separate charge isomers caused by different isoelectric points of proteins and inconsistent space charge distribution at the same time, but the mutual separation effect of the ion exchange chromatography on different proteins is uncertain due to the limitation of exchange chromatography media and the complexity of optimization of various experimental parameters.
Because the structure of the recombinant bispecific antibody drug changes in complexity and diversity in the industrial production process, the detection and analysis method which has high sensitivity and strong specificity and can detect the charge heterogeneity and homodimer impurity of the recombinant bispecific antibody drug simultaneously is an effective means for controlling and stabilizing the production quality of the recombinant protein drug.
Disclosure of Invention
The invention mainly solves the technical problem of providing a method for detecting impurities and charge variants capable of specifically binding GPC3 and CD3 protein homodimers. The high performance liquid-ion exchange chromatography can be suitable for analyzing the charge variant which has a complex structure and can specifically bind GPC3 and CD3 proteins, solves the problems that the sample is denatured due to reagent or pretreatment, and the separation of charge isomers cannot be realized by the traditional high-salt elution-ion exchange chromatography, and the like, can be used in the field of quality control of medicaments, ensures the batch-to-batch consistency of medicament production, and monitors the safety and the effectiveness of clinical medicaments.
In order to solve the technical problems, the invention adopts the following technical scheme:
the present invention provides a method for detecting the content of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins, comprising the steps of:
analyzing the protein to be detected by high performance liquid chromatography-ion exchange chromatography to obtain a chromatogram, and determining the content of impurities and charge variants of the homodimers of GPC3 and CD3 proteins which can be specifically combined to be detected based on the chromatogram;
the mobile phase adopted by the high performance liquid chromatography-ion exchange chromatography comprises a mixed solution of phosphate; forming a mobile phase A and a mobile phase B through different mixing ratios;
in the above method, preferably, the mobile phase a is a disodium hydrogen phosphate solution, and the mobile phase B is a sodium dihydrogen phosphate solution.
In the above method, preferably, the disodium hydrogen phosphate solution is 40mM, and the sodium dihydrogen phosphate solution is 40mM.
In the above method, preferably, the chromatographic column used for the high performance liquid chromatography-ion exchange chromatography comprises a cation exchange chromatographic column; preferably, the chromatography column comprises a strong cation exchange chromatography column SCX.
In the above method, the elution time of the high performance liquid chromatography-ion exchange chromatography is preferably 20 to 70min, and more preferably 55min.
In the above method, preferably, the loading amount of the high performance liquid chromatography-ion exchange chromatography is 10-50. Mu.L, the flow rate is 0.5-1.5ml/min, the column temperature is 20-60 ℃, and the detection wavelength is 280nm.
The elution procedure used in this method was as follows:
in another aspect, the invention also provides the use of the above-described method for detecting the amount of impurities and charge variants that specifically bind to GPC3 and CD3 proteins homodimers in quality control of production of charge variants that specifically bind to GPC3 and CD3 proteins.
In the above application, preferably, when the detected spectrum of the specific binding GPC3 and the detected spectrum of the specific binding CD3 protein are consistent with the detected spectrum of the physicochemical reference substance of the specific binding GPC3 and the detected spectrum of the specific binding CD3 protein, the activity of the biological products of the specific binding GPC3 and the specific binding CD3 protein is not affected or is at a safe level, and the biological products can be produced and released; when the spectrum of the charge variant in the charge variant capable of specifically binding to GPC3 and CD3 protein is detected to be inconsistent with the spectrum of the physicochemical control substance capable of specifically binding to GPC3 and CD3 protein charge variant, the activity of the biological product capable of specifically binding to GPC3 and CD3 protein charge variant is affected or affected to be at an unsafe level, and the process for producing the biological product needs to be regulated and improved until the charge variant of the biological product produced is at a safe level, and the biological product can not be released.
The above-mentioned "coincidence" means that the retention time of the liquid phase spectrogram is the same and the number of peaks is the same; "inconsistent" means that the liquid spectra have different retention times and different peak numbers.
In another aspect, the invention also provides the use of the above method for detecting impurities and charge variants that specifically bind GPC3 and CD3 protein homodimers.
According to the invention, through selecting proper experimental conditions, the effective separation of the components such as the target heterodimer, homodimer impurities and the like in the protein sample to be analyzed is realized;
the invention can also determine the relative content of each component in the map;
the invention aims at detecting the recombinant bispecific antibody drug homodimer impurity, and can achieve the purpose of controlling the drug quality.
Drawings
FIG. 1 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants that specifically bind GPC3 and CD3 protein homodimers in example 3 of the present invention;
FIG. 2 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants (CD 3 added) that specifically bind GPC3 and CD3 protein homodimers in example 3 of the present invention;
FIG. 3 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 1 of the present invention;
FIG. 4 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 2 of the present invention;
FIG. 5 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers of GPC3 and CD3 proteins that specifically bind in comparative example 3 of the present invention;
FIG. 6 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 4 of the present invention;
FIG. 7 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 5 of the present invention;
FIG. 8 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 6 of the present invention;
FIG. 9 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 7 of the present invention;
FIG. 10 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 8 of the present invention;
FIG. 11 is a chromatogram of high performance liquid-ion exchange chromatography detection of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins in comparative example 9 of the present invention.
Detailed Description
The invention is further illustrated by the following examples. It should be understood that the embodiments of the present invention are merely illustrative of the present invention and not limiting of the present invention, and that simple alternatives or modifications thereof, which are contemplated by the present invention, fall within the scope of the invention as claimed.
In the following examples and comparative examples, the reagents or apparatus used were conventional products commercially available without identifying the manufacturer.
Example 1 preparation of antibodies
And (3) taking out the 105-102-7 cell strain from liquid nitrogen, rapidly thawing in a 37 ℃ water bath, transferring the cells into a shake flask for culture, and wherein the cell inoculation density is 0.4-1.2X106 cells/ml.
Cells were expanded in shake flasks and passaged every 2 days. Shake flasks of different specifications were changed according to culture volumes, shaking parameters were 37 ℃, 5% CO2, 115rpm. When the final cell density reaches 3-6×106 cells/ml, the cell activity is more than 90%, and transferring the cells into a fermentation tank for culture.
The inoculation density of the fermentation tank is 0.8-1.2X106 cells/ml, the temperature is 37 ℃, the rotating speed is 180-250 rpm, the pH is 6.8-7.2, and the dissolved oxygen (pO 2) is 50%. When the cell density was not less than 10X 106 cells/ml, the culture temperature was lowered to 32 ℃. To maintain the cells at a high rate of activity and produce the desired protein, the initial culture volume of 3% of the feed medium was supplemented on days 4, 6, 8, and 10 of the culture. To avoid significant effects of too low glucose levels on cell viability, a 30% (m/v) glucose solution was added to the reactor to maintain the glucose level in the broth at 4g/L. And when the cell viability is reduced to about 80%, harvesting the cell fermentation broth.
The obtained fermentation broth was centrifuged AT 8000-12000 rpm for 30 minutes, and after filtration through a 0.45um filter membrane, protein A affinity chromatography (Bogurone AT protein A) was performed to obtain bispecific antibodies against GPC3 and CD3 (homodimer impurity 15% or so).
Example 2
The present embodiment provides a method for preparing a reagent material, which specifically comprises the following steps:
(1) Mobile phase a:40mM disodium hydrogen phosphate solution: accurately weighing Na2HPO4.12H2O_14.33 g, adding about 800ml of ultrapure water, and adding water to 1L after complete dissolution.
(2) Mobile phase B:40mM sodium dihydrogen phosphate solution: naH2 HPO4.2H2O_12.48 g was precisely weighed, about 1800ml of ultrapure water was added thereto, and after complete dissolution, water was added thereto to 2L.
(3) Reference solution: and (3) taking a reference substance, diluting with water to a protein concentration of about 3mg/ml according to the protein marked amount, filtering with a 0.22 mu m filter membrane, and taking filtrate as a reference substance solution.
(4) Test solution: and (3) taking a sample, diluting the sample with water to a protein concentration of about 3mg/ml according to the protein labeled amount, filtering the sample with a 0.22 mu m filter membrane, and taking filtrate for sample injection.
Example 3
The present embodiment provides a method for detecting the content of impurities and charge variants capable of specifically binding GPC3 and CD3 protein homodimers by high performance liquid chromatography-ion exchange chromatography, specifically comprising the following steps:
the chromatographic conditions are shown in table 1:
TABLE 1
| Conditional content | Name/index |
| Instrument for measuring and controlling the intensity of light | Liquid chromatograph |
| Detector/wavelength | DAD/280nm |
| Chromatographic column | ProPac Elite WCX 5um |
| Flow rate | 0.8ml/min |
| Concentration of sample | 3㎎/ml |
| Sample loading amount | 10μL |
| Column temperature | 40℃ |
The corresponding solvents were configured as in example 1. Through repeated experiments, the experimental conditions are repeatedly adjusted to obtain a proper elution gradient, as shown in table 2:
TABLE 2
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 8 | 92 |
| 4 | 0.8 | 8 | 92 |
| 14 | 0.8 | 43.5 | 56.5 |
| 15 | 0.8 | 55 | 45 |
| 20 | 0.8 | 61 | 39 |
| 30 | 0.8 | 81 | 19 |
| 36 | 0.8 | 92 | 8 |
| 41 | 0.8 | 92 | 8 |
| 47 | 0.8 | 8 | 92 |
| 55 | 0.8 | 8 | 92 |
The experiment was performed by adding a control group of CD3 antibody, and the result is shown in FIGS. 1 and 2, and the separation effect is good, and homodimer and charge heterogeneity can be simultaneously analyzed, so that the chromatographic condition of example 2 was selected as an analytical method for homodimer and charge heterogeneity capable of specifically binding GPC3 and CD3 proteins.
Comparative example 1
Chromatographic column: proPac WCX-10
In this embodiment, a set of elution gradients are designed according to the difference of isoelectric points, as shown in table 3:
TABLE 3 Table 3
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 100 | 0 |
| 4 | 0.8 | 100 | 0 |
| 15 | 0.8 | 60 | 40 |
| 15.1 | 0.8 | 60 | 40 |
| 28 | 0.8 | 60 | 40 |
| 28.1 | 0.8 | 100 | 0 |
| 32 | 0.8 | 100 | 0 |
The resulting chromatogram is shown in FIG. 3. The results show improved baseline drift conditions and separation; however, the main peak charge heterogeneity analysis was not achieved.
Comparative example 2
Chromatographic column: proPac Elite WCX 5um
In order to solve the problem of comparative example 1, the chromatographic column more suitable for separating homodimer was replaced with the recommended mobile phase, and the elution gradient was set according to the isoelectric point of the sample, and the chromatogram was observed. Elution gradients are shown in table 4:
TABLE 4 Table 4
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 100 | 0 |
| 4 | 0.8 | 100 | 0 |
| 15 | 0.8 | 80 | 20 |
| 16 | 0.8 | 80 | 20 |
| 16.1 | 0.8 | 100 | 0 |
| 23 | 0.8 | 100 | 0 |
As a result, as shown in FIG. 4, the main peak of the sample was more effective than that of comparative example 1, but the homodimer was not distinguishable, and in the case of the same amount of sample, the peak height was severely lowered, and it was presumed that the elution was insufficient and there was no eluted substance.
Comparative example 3
Chromatographic column: proPac Elite WCX 5um
The elution gradient was moderately adjusted according to the chromatogram and chromatographic conditions of comparative example 2, and the elution time was increased, as shown in table 5:
TABLE 5
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 100 | 0 |
| 4 | 0.8 | 100 | 0 |
| 20 | 0.8 | 50 | 50 |
| 30 | 0.8 | 50 | 50 |
| 30.1 | 0.8 | 100 | 0 |
| 36 | 0.8 | 100 | 0 |
As a result, as shown in FIG. 5, a new peak appears at 2min, and GPC3 may be eluted.
Comparative example 4
Chromatographic column: proPac Elite WCX 5um
In order to further improve the elution effect, the elution gradient is continuously moderately adjusted. Elution gradients are shown in table 6:
TABLE 6
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 100 | 0 |
| 4 | 0.8 | 100 | 0 |
| 14 | 0.8 | 90 | 10 |
| 30 | 0.8 | 40 | 60 |
| 40 | 0.8 | 40 | 60 |
| 40.1 | 0.8 | 100 | 0 |
| 46 | 0.8 | 100 | 0 |
As a result, as shown in FIG. 6, the peak at 20min was GPC3, and the degree of separation from the main peak was satisfactory.
Comparative example 5
Chromatographic column: proPac Elite WCX 5um
And (3) trying to finely adjust the gradient to enhance the separation effect of the main peak acid and alkali peaks, and observing whether homodimers and main peak charge heterogeneity can be achieved simultaneously in the same analysis method. Elution gradients are shown in table 7:
TABLE 7
As shown in FIG. 7, the degree of separation of the homodimer from the main peak was as desired, and the effect of separation of the main peak from the acid-base peak was as desired.
Comparative example 6
Chromatographic column: proPac Elite WCX 5um
And then the elution time of the main peak acid-base peak is increased to observe whether the better separation effect exists. The specific elution gradients are shown in table 8:
TABLE 8
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 100 | 0 |
| 4 | 0.8 | 100 | 0 |
| 5 | 0.8 | 90 | 10 |
| 15 | 0.8 | 75 | 25 |
| 16 | 0.8 | 60 | 40 |
| 40 | 0.8 | 40 | 60 |
| 41 | 0.8 | 100 | 0 |
| 42 | 0.8 | 100 | 0 |
As a result, as shown in FIG. 8, the separation effect was not significantly different from that of the previous method, and therefore, the chromatographic condition of comparative example 6 was temporarily set as the homodimer and main peak charge heterogeneity analysis method.
Comparative example 7
Chromatographic column: proPac Elite WCX 5um
The homodimer GPC3 peak often appears before the retention time is not fixed, the peak appears at a high and narrow level, and at the same time, the GPC3 peak becomes small or disappears, and it is hypothesized that under the current chromatographic conditions, GPC3 has the risk of not being combined with the chromatographic column packing, so that the homology and specific analysis result are unstable, and therefore the elution gradient needs to be adjusted, so that GPC3 and the chromatographic column are fully combined, and the separation purpose is achieved. Specific elution concentrations are shown in table 9:
TABLE 9
As a result, as shown in FIG. 9, a peak appears at 3min, and it is assumed that the difference in the charge of GPC3 at 8min is also a result of some GPC3.
Comparative example 8
Chromatographic column: proPac Elite WCX 5um
On the basis of comparative example 7, the elution gradient was further adjusted as shown in table 10:
table 10
| Time/min | Flow rate/(ml/min) | ％A(Na2HPO4.12H2O) | ％B(NaH2PO4.2H2O) |
| 0.01 | 0.8 | 15 | 85 |
| 4 | 0.8 | 15 | 85 |
| 5 | 0.8 | 22.5 | 77.5 |
| 11 | 0.8 | 43.5 | 56.5 |
| 12 | 0.8 | 55 | 45 |
| 17 | 0.8 | 61 | 39 |
| 22 | 0.8 | 81 | 19 |
| 23 | 0.8 | 81 | 19 |
| 28 | 0.8 | 15 | 85 |
| 29 | 0.8 | 15 | 85 |
As a result, as shown in FIG. 10, the peak at 11min was GPC3, and no other peak was found before, which was a very large improvement over the previous chromatographic conditions.
Comparative example 9
Chromatographic column: proPac WCX-10
The elution time was 23min. The elution procedure is shown in table 11:
TABLE 11
The resulting chromatogram is shown in FIG. 11. The result shows that the chromatogram has a main peak and an impurity peak, and the integral separation degree can meet the requirement; the gradient change is too large, and the baseline drift amplitude is too large; the main peak of the acid-base peak does not achieve the separation purpose. The impurity peaks before the main peak were presumed to be GPC 3-Mabs, but under the same chromatographic conditions, CD 3-Mabs were not retained, and the purpose of analyzing homodimers could not be achieved.
Claims (4)
1. A method of detecting the content of impurities and charge variants that specifically bind GPC3 and CD3 protein homodimers, comprising the steps of:
(1) Analyzing the protein to be detected by high performance liquid chromatography-ion exchange chromatography to obtain a chromatogram, and determining the content of impurities and charge variants of the homodimers of GPC3 and CD3 proteins which can be specifically combined to be detected based on the chromatogram;
(2) The mobile phase adopted by the high performance liquid chromatography-ion exchange chromatography comprises a mixed solution of phosphate; the preparation method comprises the steps of forming a mobile phase A and a mobile phase B through different mixing ratios, wherein the mobile phase A adopts a disodium hydrogen phosphate solution, the mobile phase B adopts a sodium dihydrogen phosphate solution, the disodium hydrogen phosphate solution is 40mM, and the sodium dihydrogen phosphate solution is 40mM;
(3) The high performance liquid chromatography-ion exchange chromatography adopts a chromatographic column, wherein the chromatographic column is an ion exchange chromatographic column WCX;
(4) The loading amount of the high performance liquid chromatography-ion exchange chromatography is 10 mu L, the loading concentration is 3mg/mL, the flow rate is 0.8mL/min, the column temperature is 40 ℃, and the detection wavelength is 280nm;
wherein the elution procedure employed in the method is as follows:
。
2. Use of the method of detecting the content of impurities and charge variants of homodimers capable of specifically binding GPC3 and CD3 proteins according to claim 1 for the quality control of the production of charge variants capable of specifically binding GPC3 and CD3 proteins.
3. The use according to claim 2, wherein the detected spectrum of the charge variant in the charge variant of the specifically binding GPC3 and CD3 protein is consistent with the spectrum of the physicochemical control of the charge variant of the specifically binding GPC3 and CD3 protein, and the activity of the resulting biologic of the specifically binding GPC3 and CD3 protein charge variant is not affected or is at a safe level, allowing for production release; when the spectrum of the charge variant in the charge variant capable of specifically binding to GPC3 and CD3 protein is detected to be inconsistent with the spectrum of the physicochemical control substance capable of specifically binding to GPC3 and CD3 protein charge variant, the activity of the biological product capable of specifically binding to GPC3 and CD3 protein charge variant is affected or affected to be at an unsafe level, and the process for producing the biological product needs to be regulated and improved until the charge variant of the biological product produced is at a safe level, and the biological product can not be released.
4. Use of the method of claim 1 for detecting impurities and charge variants which bind specifically to GPC3 and CD3 protein homodimers.
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| CN111175404A (en) * | 2020-01-19 | 2020-05-19 | 东莞市东阳光生物药研发有限公司 | High performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drug |
| CN111239278A (en) * | 2020-02-05 | 2020-06-05 | 康立泰药业有限公司 | Method for detecting recombinant human interleukin-12 protein charge variant and application |
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| CN111175404A (en) * | 2020-01-19 | 2020-05-19 | 东莞市东阳光生物药研发有限公司 | High performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drug |
| CN111239278A (en) * | 2020-02-05 | 2020-06-05 | 康立泰药业有限公司 | Method for detecting recombinant human interleukin-12 protein charge variant and application |
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