CN114740108B - Method for measuring modification degree of polymer modified antibody drug - Google Patents
Method for measuring modification degree of polymer modified antibody drug Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a method for measuring the modification degree of a polymer modified antibody drug, in particular to a method for measuring the modification degree of a PEG modified monoclonal antibody. According to the determination method, the degree of modification of the polymer (such as PEG) on the monoclonal antibody chain is calculated by utilizing LC-Q-TOF, complete physical separation or chromatographic separation of the monoclonal antibody chains with different degrees of modification is not needed, and the degree of modification of the polymer (such as PEG) on the light chain and the heavy chain can be obtained simultaneously by utilizing a deconvolution molecular weight difference rule; moreover, without physical separation, molecular weight detection is carried out on up to hundreds of thousands of polymer modified antibodies by MALDI-TOF, and molecular weight detection can also be carried out on the light chain and the heavy chain after reduction and resolution, so as to calculate the modification degree of the polymer (such as PEG); LC-Q-TOF and MALDI-TOF are combined to be used to complement each other, and mutually verify, so that more modification information of the heavy chain and the light chain can be obtained, and the result is more reliable.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for measuring the modification degree of a polymer modified antibody medicine (especially a PEG modified monoclonal antibody) and application thereof.
Background
The protein medicine mainly comprises polypeptide, enzyme, cell factor, hormone, antibody and other proteins with special functions, and along with the development of biotechnology, people can obtain the required proteins through fermentation engineering, genetic engineering, protein engineering, enzyme engineering and other approaches. However, a great number of clinical researches show that the protein medicine without any modification has the defects of short half-life period, strong immunogenicity, poor stability, easy degradation by in vivo enzymes and the like in vivo, thereby reducing the clinical effect. For this reason, attempts have been made to modify protein drugs in various ways to suit clinical applications.
Polyethylene glycol (PEG) is a free-crimping uncharged water-soluble polymer in solution, which has no toxicity, weak antigenicity and good biocompatibility, and covalent modification of proteins can increase the in vivo circulation half-life of proteins and reduce antigenicity, increase the solubility of proteins and change the biological distribution of proteins in human body. Since Abuchowski, davis (J.biol. Chem.1977,252: 3578-3581.) et al reported modification of proteins with PEG for the first time in 1977, PEG has been widely used in the modification studies of protein polypeptides, and protein PEGylation technology has become one of the most effective methods for reducing the immunogenicity and improving the pharmacokinetic/pharmacodynamic properties of protein biopharmaceuticals. In recent years, PEG has also been used as a linker for the preparation of antibody Drug conjugates (Antibody Drug Conjugates, ADC) consisting of three parts, antibody (anti), drug (Drug) and linker (linker). The antibody drug conjugate can realize 'accurate treatment' due to good targeting and anti-tumor activity, and has become a new hot spot and important trend for developing anti-tumor antibody drugs, and is receiving more and more attention.
Because the protein has more sites for modification, the average modification degree of the PEG on the protein modification degree or the PEG combination number is reflected, and the method has important significance for researching the structure-activity relationship of the drugs and controlling the quality. At present, the detection method of the average modification degree of PEG mainly comprises the following steps: trinitrobenzenesulfonic acid (TTBS) method, fluorescamine method, mass spectrometry, liquid chromatography, capillary method, fourier Transform Infrared (FTIR) spectroscopy, raman scattering spectroscopy, enzymolysis method, etc. In practical application, the TTBS method and the fluorescamine method are found to be simple and easy to operate, but are greatly interfered by the outside, so that larger experimental errors are caused, for example, as described in a comparative example of patent document CN 108267590A. CN108267590a discloses a method for detecting PEG-binding number of PEG-modified protein, which uses enzymolysis method to treat the PEG-modified protein sample to be detected, then releases PEG, detects the quality of PEG, and calculates the PEG-binding number in the PEG-modified protein through the mole ratio of protein and PEG. However, the enzymatic hydrolysis method may release incomplete PEG, and in order to avoid interference of free PEG, purification of the enzymatic hydrolysis sample is often required. Patent document CN109682901a discloses a method for determining average modification degree of polyethylene glycol protein drugs, which comprises the steps of using a plurality of types of detectors which are in linear relation with the concentration of an unmodified protein drug standard substance and the concentration of a polyethylene glycol standard substance, respectively establishing standard curves of the response values of each type of detector corresponding to the concentration of the unmodified protein drug standard substance and the concentration of the polyethylene glycol standard substance and the respective concentrations, wherein the steps are complicated, and the workload in actual detection is large.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for measuring the modification degree of a polymer modified protein drug (particularly a PEG modified monoclonal antibody) and application thereof.
In a first aspect of the present invention, there is provided a method for determining the degree of modification of a polymer modified antibody, comprising the steps of detecting a sample to be detected by MALDI-TOF, treating the sample to be detected with a reducing agent, and detecting the sample by LC-Q-TOF and MALDI-TOF, respectively.
Specifically, the steps of detecting the sample to be detected through LC-Q-TOF and MALDI-TOF after being treated by the reducing agent respectively comprise the following steps:
(1) Mixing a sample to be tested with a reducing agent, and carrying out a reduction reaction;
(2) And (3) detecting the product obtained in the step (1) through LC-Q-TOF and MALDI-TOF respectively.
Optionally, the method further comprises the step of mutually verifying the obtained LC-Q-TOF detection result and MALDI-TOF detection result.
Specifically, if necessary, the sample to be measured may be diluted and mixed with a reducing agent, which may be a buffer solution such as NH 4 HCO 3 A solution.
Specifically, the reducing agent may be selected from: beta-mercaptoethanol, tris (2-carboxyethyl) phosphorus, dithiothreitol (DTT), and in particular DTT.
In one embodiment of the invention, the reducing agent is DTT at a concentration of 100-1000mmol/L (e.g., 100, 200, 300, 400, 500, 600, 800, 1000 mmol/L).
In particular, the temperature of the reduction reaction in step (1) is from 35 to 40 ℃ (e.g. 35, 36, 37, 38, 39, 40 ℃), in particular 37 ℃.
Specifically, the time of the reduction reaction in step (1) is 10 to 30 minutes (e.g., 10, 15, 20, 25, 30 minutes), particularly 20 minutes.
Specifically, the reduction reaction in step (1) is performed under shaking conditions.
Specifically, the matrix of MALDI-TOF is 3, 5-dimethoxy-4-hydroxycinnamic acid (sinapic acid, SA).
Specifically, the Power of the MALDI-TOF is 150.
In one embodiment of the invention, the instrument used for MALDI-TOF detection is MALDI-8020.
Specifically, the LC-Q-TOF is UPLC-Q-TOF, the stationary phase of the liquid chromatography may be UPLC BEH C18, and the particle size thereof may be 1-5 μm, for example 1.7 μm.
In particular, the column temperature of the liquid chromatography may be from 30 to 50 ℃ (e.g. 30, 35, 36, 38, 40, 42, 44, 45, 50 ℃), in particular from 35 to 45 ℃, e.g. 40 ℃.
In particular, the mobile phase flow rate of the liquid chromatography may be in the range of 0.1-1mL/min (e.g. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mL/min), in particular 0.1-0.5mL/min, e.g. 0.2mL/min.
Specifically, the mobile phase is composed of a mobile phase aqueous phase (a) and a mobile phase organic phase (B).
In particular, mobile phase A is an aqueous formic acid solution having a concentration of 0.01 to 1% (volume percent) (e.g., 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.4%, 0.5%, 1%), in particular 0.05 to 0.2%, e.g., 0.1%).
In particular, mobile phase B is an acetonitrile solution of formic acid at a concentration of 0.01 to 1% (volume percent) (e.g., 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.4%, 0.5%, 1%), particularly 0.05 to 0.2%, e.g., 0.1%.
Specifically, the liquid chromatography detection employs gradient elution; more specifically, the elution procedure includes:
0-1min, mobile phase B,5%, mobile phase A, and the balance;
1-30min, mobile phase B,5% -25%, mobile phase A and the balance;
30-40min, mobile phase B,25% -50%, mobile phase A and the balance;
40-50min, mobile phase B,55% -75%, mobile phase A, and the rest;
50-60min, mobile phase B,75% -100%;
60-60.1min, mobile phase B,100% -5%, mobile phase A and the balance;
60.1-70min, mobile phase B, 5%. Fwdarw. Stop, mobile phase A, balance.
Specifically, the liquid chromatography is performed at a sample loading of 1 to 5. Mu.L (e.g., 1, 2, 3, 4, 5. Mu.L), and particularly 4. Mu.L.
Specifically, for the LC-Q-TOF, the ion source of the mass spectrum is ESI, ionization mode: positive ions.
In particular, for the LC-Q-TOF, the mass spectrum has a scanning mass in the range of 400-3000m/z.
In particular, for the LC-Q-TOF, the declustering voltage (DP) of the mass spectrum is 250V.
Specifically, for the LC-Q-TOF, the collision voltage (CE) of the mass spectrum is 10V.
Specifically, for the LC-Q-TOF, accumulation time of the mass spectrum is 0.15 seconds.
Specifically, for the LC-Q-TOF, the Time bin to sum of the mass spectrum is 80.
In one embodiment of the invention, the mass spectrometry instrument is a TripleTOF 6600.
Specifically, the sample to be tested includes a polymer modified antibody sample and an antibody sample.
Specifically, the polymer in the polymer modified antibody is a water-soluble polymer, such as polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyoxazoline, polyacrylamide morpholine, polysialic acid, dextran, and the like; in one embodiment of the present invention, the polymer is polyethylene glycol (PEG), and the method is a method for determining the PEG binding number of a PEG-modified antibody.
In particular, the molecular weight of the polymer is 400-100000 daltons (e.g. 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, 50000, 100000 daltons), in particular 400-4000 daltons.
In particular, the polyethylene glycol is a linear or branched polyethylene glycol, in particular a linear polyethylene glycol.
In particular, the polyethylene glycol comprises a single-ended, double-ended or multi-ended activated activating group, in particular a single-ended activated activating group; more specifically, the activating groups include, but are not limited to, aldehyde groups, carboxyl groups, ester groups, amide groups, mercapto groups, epoxy groups, maleimide groups, succinimidyl carbonates, succinimidyl acetates, succinimidyl propionates, carbonyl aldehydes, hydrazides, amino groups, and the like.
Specifically, the antibodies in the polymer modified antibodies are monoclonal antibodies; in one embodiment of the invention, the antibody is EGFR mab, e.g., rituximab (Necitumumab).
Specifically, the method comprises an assay step of detecting the degree of modification on the light chain and/or heavy chain of a monoclonal antibody.
Specifically, the method is a method for determining the degree of modification on the light and heavy chains of a PEG-modified monoclonal antibody.
In one embodiment of the invention, the PEG-modified monoclonal antibody N3- (CH) 2 CH 2 O) n CH 2 CH 2 CO-monoclonal antibody, n is the degree of polymerization; in one embodiment of the invention, the PEG-modified monoclonal antibody is N3- (CH) 2 CH 2 O) 12 CH 2 CH 2 CO-rituximab.
In particular, the polymer modified antibodies may be Antibody Drug Conjugates (ADCs) in which the polymer (e.g., PEG) moiety acts as a linker.
In a second aspect of the invention there is provided the use of the method of the first aspect in quality control of a polymer modified antibody.
In particular, the polymer, antibody has the definition set forth in the first aspect of the invention.
In particular, the use of the method of the first aspect in the determination of the PEG binding number of a PEG-modified antibody.
In one embodiment of the invention, the antibody is EGFR mab, e.g., rituximab (Necitumumab).
In one embodiment of the invention, the PEG-modified monoclonal antibody is N3- (CH) 2 CH 2 O) n CH 2 CH 2 CO-monoclonal antibody, n is the degree of polymerization; in one embodiment of the invention, the PEG-modified monoclonal antibody is N3- (CH) 2 CH 2 O) 12 CH 2 CH 2 CO-rituximab.
In particular, the polymer modified antibodies may be Antibody Drug Conjugates (ADCs) in which the polymer (e.g., PEG) moiety acts as a linker.
According to the determination method provided by the invention, on one hand, the modification degree of the polymer (such as PEG) on the monoclonal antibody chain is calculated by utilizing the LC-Q-TOF, complete physical separation or chromatographic separation of the monoclonal antibody chains with different modification degrees is not needed, and the modification degrees of the polymer (such as PEG) on the light chain and the heavy chain can be obtained simultaneously by utilizing the deconvolution molecular weight difference rule; on the other hand, without physical separation, molecular weight detection can be carried out on up to hundreds of thousands of polymer modified antibodies (such as PEG modified antibodies) by MALDI-TOF, and molecular weight detection can also be carried out on the light chain and the heavy chain after reduction and resolution, thus the modification degree of the polymer (such as PEG) on the light chain and the heavy chain can be obtained simultaneously; LC-Q-TOF and MALDI-TOF are combined to be used to complement each other, and mutually verify, so that more modification information of the heavy chain and the light chain can be obtained, and the result is more reliable. Compared with the prior art, the measuring method provided by the invention is simple, quick and high in accuracy.
Drawings
Fig. 1 shows a total ion flow diagram of EGFR mab after DTT treatment.
Figure 2 shows the deconvolution profile of the EGFR mab light chain.
FIG. 3 shows a deconvolution profile of EGFR monoclonal antibody heavy chains.
FIG. 4 shows the total ion flow diagram of EGFR-PEG after DTT treatment.
FIG. 5 shows a deconvolution profile of EGFR-PEG light chains.
FIG. 6 shows a deconvolution profile of EGFR-PEG heavy chains.
FIG. 7 shows MALDI-TOF spectra of EGFR mab.
FIG. 8 shows MALDI-TOF spectra of EGFR-PEG.
FIG. 9 shows MALDI-TOF spectra of light chain of EGFR mab after DTT treatment.
FIG. 10 shows MALDI-TOF spectra of light chain of EGFR-PEG treated with DTT.
FIG. 11 shows MALDI-TOF spectra of heavy chains of EGFR mab after DTT treatment.
FIG. 12 shows MALDI-TOF spectra of heavy chains of EGFR-PEG after DTT treatment.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.
Various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
With EGFR-PEG (N3)(CH 2 CH 2 O) 12 CH 2 CH 2 CO-EGFR mab) as an example, the detection method of the present invention and its detection effect are illustrated.
EGFR mab is rituximab (Necitumumab), available from Eli Lilly corporation.
The EGFR-PEG was prepared as follows: according to EGFR monoclonal antibody (mAb hereinafter) and N3-PEG 12 The reaction was carried out with a feed ratio of 1:20 of SPA (supplied by Beijing key Kai technologies Co., ltd.). mu.L of mAb solution (concentration: 9.6 mg/mL) was first weighed and placed in a 1.5mL centrifuge tube, and then the mAb was weighed to be 2mg. 10mg/mL of N3-PEG was formulated using 5mM PBS solution at pH3.0 12 SPA solution, 20. Mu.L was weighed into a centrifuge tube, mixed well and reacted for 2h with shaking. Removing unreacted N3-PEG by ultrafiltration after the reaction is finished 12 -SPA。
EGFR mab and EGFR-PEG samples were analyzed on-board by MALDI-TOF.
EGFR-PEG 1mg/mL, EGFR-PEG 50. Mu.L plus 200mM DTT solution 2.5. Mu.L, shaking at 37℃for 20 minutes, and samples were analyzed by LC-Q-TOF and MALDI-TOF, respectively, on-machine.
The EGFR monoclonal antibody pure product is detected and analyzed under the same condition.
The relevant parameters are as follows:
Q-TOF：SCIEX TripleTOF 6600
MALDI-TOF：SHIMADZU MALDI-8020
table 1 instrument and parameters
The results are shown in FIGS. 1-12.
LC-Q-TOF can deconvolute the total ion flow diagrams (figure 1) of the light chain and the heavy chain of EGFR monoclonal antibody after DTT treatment to obtain the molecular weight of the light chain (figure 2) and the molecular weight of the heavy chain (figure 3); the light chain mixture and the heavy chain mixture with different PEG modification degrees after the EGFR-PEG conjugate is subjected to DTT treatment do not need to be completely separated, the light chain mixture and the heavy chain mixture are directly deconvolved on a total ion flow diagram (shown in figure 4), and the main range of the light chain and heavy chain PEG modification degrees can be obtained according to the difference rule of mass numbers and is 1-3 (shown in figures 5 and 6);
MALDI-TOF is directly carried out on EGFR monoclonal antibody and EGFR-PEG respectively, the integral PEG modification degree of the EGFR monoclonal antibody is about 9 (see fig. 7 and 8) according to the mass number difference (152388-146955)/626=8.7), the spectrogram on LC-Q-TOF is complex due to the larger molecular weight of EGFR monoclonal antibody and EGFR-PEG, the PEG modification number is difficult to solve, and MALDI-TOF can be supplemented in the aspect;
MALDI-TOF is respectively carried out on the light chain of EGFR monoclonal antibody and EGFR-PEG after DTT treatment, the main range of PEG modification degree of the EGFR monoclonal antibody light chain is calculated to be 1-3 according to the mass number difference (see figure 9 and figure 10), the PEG modification degree is consistent with the LC-Q-TOF analysis result, and the EGFR monoclonal antibody light chain and the EGFR-PEG are mutually verified;
MALDI-TOF is respectively carried out on heavy chains of EGFR monoclonal antibody and EGFR-PEG after DTT treatment, the number of modification of the EGFR heavy chains is calculated to be 3 according to the mass number difference (see fig. 11 and 12), and LC-Q-TOF can provide more details in the aspect of PEG modification of the heavy chains, so that the defect of less information amount of MALDI can be overcome.
From the above examples, it can be seen that LC-Q-TOF and MALDI-TOF are both complementary and mutually validated, and provide detailed resolution of the degree of modification of EGFR monoclonal antibody conjugated PEG.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.
The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.
Claims (20)
1. A method for determining the degree of modification of a polymer-modified antibody, comprising the steps of: the polymer modified antibody sample and the antibody sample are detected by MALDI-TOF respectively, and the integral modification degree is obtained according to the mass number difference; after the antibody sample is treated by a reducing agent, the total ion flow is deconvoluted through LC-Q-TOF detection, and the molecular weights of the light chain and the heavy chain of the antibody are obtained; after a polymer modified antibody sample is treated by a reducing agent, deconvolution is carried out on the total ion flow through LC-Q-TOF detection, so as to obtain the modification degree of the polymer modified antibody on the light chain and the heavy chain; the light chain and the heavy chain of the polymer modified antibody sample and the antibody sample treated by the reducing agent are detected by MALDI-TOF respectively, so as to obtain the modification degree of the light chain and the heavy chain; mutually verifying the MALDI-TOF detection result and the LC-Q-TOF detection result;
the polymer is polyethylene glycol, and the antibody is EGFR monoclonal antibody;
the reducing agent is selected from: beta-mercaptoethanol, tris (2-carboxyethyl) phosphorus, dithiothreitol, or combinations of one or more thereof.
2. The method of claim 1, wherein the reducing agent is dithiothreitol.
3. The method of claim 1, wherein the temperature of the treatment is 35-40 ℃; and/or the number of the groups of groups,
the treatment time is 10-30 minutes.
4. The method of claim 1, wherein the temperature of the treatment is 37 ℃.
5. The method of claim 1, wherein the matrix of MALDI-TOF is 3, 5-dimethoxy-4-hydroxycinnamic acid; and/or, the Power of the MALDI-TOF is 150.
6. The method of claim 1, wherein the LC-Q-TOF is UPLC-Q-TOF and the stationary phase of LC is UPLC BEH C18.
7. The method of claim 1, wherein the LC has a column temperature of 30-50 ℃.
8. The method of claim 1, wherein the LC has a column temperature of 35-45 ℃.
9. The method of claim 1, wherein the LC has a mobile phase flow rate of 0.1-1mL/min.
10. The method of claim 1, wherein the mobile phase consists of a mobile phase aqueous phase a and a mobile phase organic phase B, the mobile phase a is an aqueous formic acid solution, wherein the volume percentage concentration of formic acid is 0.01-1%, and the mobile phase B is an acetonitrile solution of formic acid, wherein the volume percentage concentration of formic acid is 0.01-1%.
11. The method of claim 10, wherein the LC detection employs gradient elution, and wherein the elution procedure comprises:
0-1min, mobile phase B,5%, mobile phase A, and the balance;
1-30min, mobile phase B,5% -25%, mobile phase A and the balance;
30-40min, mobile phase B,25% -50%, mobile phase A and the balance;
40-50min, mobile phase B,55% -75%, mobile phase A, and the rest;
50-60min, mobile phase B,75% -100%;
60-60.1min, mobile phase B,100% -5%, mobile phase A and the balance;
60.1-70min, mobile phase B, 5%. Fwdarw. Stop, mobile phase A, balance.
12. The method of claim 1, wherein for the LC-Q-TOF, the ion source of the Q-TOF is ESI, ionization mode: positive ions, the scanning mass range of the Q-TOF is 400-3000m/z.
13. The method of claim 1, wherein for the LC-Q-TOF, the declustering voltage (DP) of the Q-TOF is 250V.
14. The method of claim 1, wherein the collision voltage (CE) of the Q-TOF is 10V for the LC-Q-TOF.
15. The method of claim 1, wherein Accumulation time of the Q-TOF is 0.15 seconds for the LC-Q-TOF.
16. The method of claim 1 wherein for the LC-Q-TOF, the Time bin to sum of the Q-TOF is 80.
17. The method of any one of claims 1-16, wherein the polymer has a molecular weight of 400 to 100000 daltons.
18. The method of any one of claims 1-16, wherein the polymer modified antibody is N 3 -(CH 2 CH 2 O) 12 CH 2 CH 2 CO-rituximab.
19. The method of any one of claims 1-16, wherein the polymer modified antibody is an antibody drug conjugate.
20. Use of the method of any one of claims 1-19 for quality control of a polymer modified antibody.
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