CN103217499A - Method for determining immunoglobulin charge isomer glycosylation and terminal modification states - Google Patents
Method for determining immunoglobulin charge isomer glycosylation and terminal modification states Download PDFInfo
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Abstract
The invention provides a method for determining immunoglobulin charge isomer glycosylation and terminal modification states. With the method, immunoglobulin glycosylation, N-terminal pyroglutamic acidification, and C-terminal de-lysine can be simultaneously determined rapidly. The method comprises the steps that: (1) immunoglobulin before and after carboxypeptidase B digestion are analyzed by using cation exchange chromatography (CEX-HPLC), and different immunoglobulin charge isomers are collected according to retention times after the column; (2) the immunoglobulin component in the step (1) is denatured by using a denaturant, and is reduced by using a reducing agent, such that light chain and heavy chain are split; (3) the light chain and heavy chain in the step (2) are separated with reversed-phase ultrahigh-pressure liquid chromatography; (4) molecular weights of the light chain and heavy chain obtained in the step (3) are determined by using mass spectrometry; and (5) the chromatographic data in the step (3) and the mass spectral data in the step (4) are analyzed, such that the glycosylation and terminal modification states of the immunoglobulin are determined.
Description
Technical field
The present invention relates to biological technical field.Particularly, the invention provides a kind of glycosylation of immunoglobulin (Ig) electric charge isomeride and method of end modified situation measured simultaneously, the invention still further relates to a kind of glycosylation of immunoglobulin (Ig) electric charge isomeride and kit of end modified situation measured simultaneously.
Background technology
Over past ten years, monoclonal antibody obtains great success and huge development in biological medicine circle and even whole pharmaceuticals industry.Advantages such as compare with traditional small-molecule drug, monoclonal antibody has high specificity, and is evident in efficacy, and spinoff is little, and consumption is few.With regard to the drug molecule characteristic, antibody has bigger inhomogeneity.This characteristic of antibody is caused that by multiple factor posttranslational modification is wherein most important internal factor.Common antibody posttranslational modification comprises glycosylation, and N holds pyroglutamic acidization, and the C end takes off lysine, deacylated tRNA amine, oxidation, isomerization etc.A plurality of links in the antibody drug research and development as Molecular Identification, process exploitation, quality monitoring etc., all need check and analysis are carried out in posttranslational modification.
The IgG antibody glycosylation occurs in the asparagine in heavy chain Fc district, belongs to the N-glycosylation, is that the important structure of antibody is formed.The core cell of IgG sugar chain is connected to form by two fork structures of two N-acetylglucosamines and three mannoses, according to the difference of terminal galactose, core fucose, terminal sialic acid etc., can form multiple different sugar chain structure.The glycosylation of IgG is inhomogenous, shows as different sugared type and content.Glycosylation difference can influence the biologic activity of antibody and medicine for feature, as removing the half life period etc. in CDC, ADCC, the body.
When the N terminal amino acid of IgG antibody is glutamine, cyclization takes place easily and generate pyroglutamic acid (pyroglutamicacid, pyroE).This reaction can spontaneously be carried out, and also can carry out under the enzymatic condition.At the C of IgG antibody molecule end, then take off lysine (K) easily.Under most of situation, the biologically active of both antagonists is influence not, but also has report to refer to that the N end pyroglutamic acidization of some antibody may influence the adhesion of itself and antigen.In addition, the glutamine pyroglutamic acidization of N end and C end take off the CHARGE DISTRIBUTION that lysine all can influence antibody, and charge characteristic is one of important indicator of antibody quality monitoring.
Therefore, it is significant in the antibody research and development to set up glycosylation of fast detecting IgG antibody and end modified analytical approach.At present, this area is generally to glycosylation with end modifiedly detect separately.The enzymolysis fluorescent marker method is the classical quantivative approach that the IgG1 glycosylation is measured, but the sample preparation process is quite complicated, length consuming time, and need sample size big; Also have the mass spectroscopy of use IgG enzymatic fragment to carry out the glycosylation analysis, as papain enzyme and IdeS enzyme etc., but these methods all come with some shortcomings, not strong as the restriction enzyme site selectivity, or cost is too high, or sample preparation complexity etc., is not suitable for the batch detection of routine or process exploitation sample.Use LC-MS and carry out glycosylation that peptide figure analysis can detect antibody in theory simultaneously and end modified, but there are many technological difficulties in the separation of high sugar content peptide, mensuration and data analysis, be not suitable for the glycosylation quantitative test, and sample preparation process complexity, length consuming time, enzyme are cut process also may original end modified the exerting an influence of antagonist.Therefore, glycosylation and the end modified report that carries out fast measuring simultaneously to the IgG that are applicable to the antibody research and development are not arranged yet at present, the method that can carry out fast, accurately characterize and identify the structure of a small amount of antibody electric charge isomeride is not arranged yet.
Summary of the invention
In order to solve the problems of the technologies described above, the purpose of this invention is to provide a kind of immunoglobulin (Ig) (the being antibody) glycosylation of electric charge isomeride and method of end modified situation measured simultaneously, this method can be simultaneously, apace immunoglobulin (Ig) glycosylation, N end pyroglutamic acidization and C end are taken off lysine measures, and can the structure of a small amount of antibody electric charge isomeride be characterized accurately and identify.Another object of the present invention provides a kind of glycosylation of immunoglobulin (Ig) electric charge isomeride and kit of end modified situation measured simultaneously.
The invention provides a kind of glycosylation of immunoglobulin (Ig) electric charge isomeride and method of end modified situation measured, said method comprising the steps of:
1) use cation-exchange chromatography (CEX-HPLC) to analyze the immunoglobulin (Ig) that the protaminase enzyme is cut front and back, and by collecting different immunoglobulin (Ig) electric charge isomeride behind the retention time post;
2) make in the step 1) immunoglobulin (Ig) after the denaturant sex change, use the reductive agent reduction, thereby split light chain and heavy chain;
3) light chain and the heavy chain of immunoglobulin (Ig) the anti-phase UHV (ultra-high voltage) liquid chromatography separating step 2 of use);
4) light chain that obtains in the use mass spectroscopy step 3) and the molecular weight of heavy chain; And
5) analytical procedure 3) in chromatographic data and the mass spectrometric data in the step 4), thereby measure the glycosylation and the end modified situation of described immunoglobulin (Ig).
Wherein, described immunoglobulin (Ig) is preferably human immunoglobulin(HIg), is preferably human immunoglobulin(HIg) IgG, more preferably human immunoglobulin(HIg) IgG1 and IgG2 hypotype.
And the glycosylation of described immunoglobulin (Ig) and end modified situation preferably include the light chain N end pyroglutamic acidization of immunoglobulin (Ig), and the asparagine glycosylation of heavy chain and N end pyroglutamic acidization, C end take off lysine.
In assay method provided by the invention, the protaminase in the described step 1) and the mass ratio of immunoglobulin (Ig) are 1: 20; The enzyme time of cutting is 3 hours.
Particularly, described step 2) comprising: the 1-6M guanidine hydrochloride aqueous solution that in a certain amount of immunoglobulin (Ig), adds 10-30 μ L, mix the back and add 1-4 μ L dithiothreitol (DTT) (DTT) aqueous solution, make immunoglobulin (Ig) generation sex change, reduction reaction, wherein, the DTT final concentration is 25-100mM in the reaction solution, and the immunoglobulin (Ig) final concentration is 0.2-3 μ g/ μ L.
Preferably, the DTT final concentration described step 2) is 50mM.
Preferably, described step 2) immunoglobulin (Ig) generation sex change, reduction reaction temperature in are 50-65 ℃, and the reaction time is 45min-120min.
More preferably, described step 2) immunoglobulin (Ig) generation sex change, reduction reaction temperature in are 65 ℃, and the reaction time is 45min.
Particularly, described step 3) specifically comprises:
Adopt reverse UHV (ultra-high voltage) liquid chromatography separating step 2) in the light chain and the heavy chain of immunoglobulin (Ig), to realize the baseline separation of described light chain and heavy chain, according to specific embodiments of the present invention, the liquid phase systems of employing can for UPLC (Waters, ACQUITY).Chromatographic column: Waters, ACQUITY UPLC column, BEH C4,1.7 μ m (particle diameter),
(aperture), 2.1 * 50mm.
The moving phase elution requirement is bigger to influencing separating of light chain and heavy chain, and preferably, chromatographic condition is set at:
Chromatogram column temperature is set at 55-65 ℃, sample size 0.2-3 μ g;
Moving phase X is 0.1% formic acid water, and moving phase Y is 0.1% formic acid acetonitrile, and flow velocity is 0.4mL/min;
Condition of gradient elution is:
Particularly, described step 4) specifically comprises:
Employing electrospray ionization mass spectrometry determination step 3) light chain that obtains in and the molecular weight of heavy chain, wherein at 0-5min, stream leads to waste liquid, 5-16min, stream leads to mass spectrum, adopts positive ion mode collection mass spectrometric data then;
Preferably, the mass spectrum condition enactment is:
The taper hole air-flow is 50.0L/Hr, and desolventizing gas is 500-800.0L/Hr, and the desolventizing temperature is 350-500 ℃, and sweep limit is 400-2500Da, and be 0.5-2s sweep time.Sampling taper hole voltage is bigger to the mass signal influence, is set at 20-40V, preferably, is set at 20-30V.
Particularly, described step 5) specifically comprises:
Calculate the N end pyroglutamic acid ratio of the light chain of described immunoglobulin (Ig) by the chromatographic peak area that obtains in the step 3), by deconvoluting calculating, the sugared type relative content and the N that obtain the heavy chain of described immunoglobulin (Ig) hold pyroglutamic acid and C end to take off lysine ratio by the mass spectrometric data that obtains in the step 4).
When the present invention carried out reduction reaction by the immunoglobulin (Ig) before and after cation-exchange chromatography (CEX-HPLC) analysis protaminase enzyme is cut, the disulfide bond of albumen ruptured under the effect of DTT, generates two the same light chains and two the same heavy chains.Use C4 anti-phase-the UHV (ultra-high voltage) liquid chromatography separates weight chain potpourri, the light chain and the heavy chain (containing different sugar type and end modified) that contain or do not contain N end pyroglutamic acid can be realized baseline separation, adopt its molecular weight of ESI-MS on-line determination then.Owing to contain the different sugar type or end modified heavy chain has different molecular weight, and the ratio of various heavy chains is directly proportional with the intensity of its molecular weight peaks, and therefore sugared type relative content and heavy chain N end pyroglutamic acidization, C end take off lysine ratio and all can be calculated by the heavy chain mass spectrometric data and obtain.In addition, light chain N end pyroglutamic acid ratio is calculated by chromatographic peak area and is obtained.
The more existing technology in this area are measured molecular weight or its modification situation of protein at present, but compare with method of the present invention, all have defective or weak point.For example mass spectrometer (MS) as MALDI-MS, is usually used in measuring the molecular weight of antibody intact proteins level.Though mass spectrometry method is good to the sample compatibility, and is easy to operate, resolution is lower as a result to record molecular weight; As the glycosylated classical quantivative approach of IgG antibody---the enzymolysis fluorescent marker method adopts N sugar acid anhydride enzyme PNGaseF enzyme to cut IgG and obtains sugar chain, carries out fluorescence labeling, high performance liquid chromatography or capillary electrophoresis analysis again after purified; This method selectivity is strong, accuracy height, but sample preparation process complexity, and length consuming time (needing 2 days usually), and need sample size big (generally being at least 100 μ g); Also have and use ESI-MS to measure the IgG enzymatic fragment to carry out the glycosylation analysis, but the selectivity of papain restriction enzyme site is low, can increase accessory substance and influence data analysis; Immunoglobulin G digestive enzyme S (IdeS) selectivity is very strong, but the cost height of IdeS, is not suitable for the batch detection of routine or process exploitation sample; Use LC-MS and carry out glycosylation that the pancreatin peptide figure analysis can detect antibody in theory simultaneously and end modified, but there are many technological difficulties in the separation of high sugar content peptide, mensuration and data analysis, and its detection sensitivity is low, be unsuitable for the detection of low content glycosyl, sample preparation process complexity in addition, length consuming time, enzyme are cut process may original end modified the exerting an influence of antagonist.
By contrast, this method adopts ESI-MS to measure the antibody multiple-charged ion, deconvoluting calculating then, improved greatly resolution and testing result accuracy (＜30ppm).In addition, sample preparation of the present invention is simple, only needs reductive agent to react (45min), amount of samples few (5 μ g), and the UPLC-MS of sample detects and only to need finish in 16min, just can obtain the data that antibody glycosylation, N end pyroglutamic acidization and C end take off lysine simultaneously.The present invention is particularly useful for the few experiment of sample size, as colony screening, and the batch fast detecting of process exploitation process; Simultaneously, the present invention also is applicable to the sample detection of conventional amount used (100 μ g).Adopt method of the present invention, structure that can also antagonist electric charge isomeride characterizes and identifies.
Immunoglobulin (Ig) can be divided into IgG, IgA, IgM, IgD, IgE five classes, and wherein IgG can be divided into hypotypes such as IgG1, IgG2, IgG3, IgG4, and the monoclonal antibody drug 70%-80% of Xiao Shouing belongs to the IgG1 albuminoid in the market.Composition according to people source amino acid sequence in the IgG antibody 1 can be divided into multiple protein such as mosaic type IgG antibody 1, humanized antibody IgG1 again.The invention provides a kind of glycosylation of immunoglobulin (Ig) electric charge isomeride and method of end modified situation measured simultaneously, a kind of glycosylation of Immunoglobulin IgG1, IgG2 and method of end modified situation measured simultaneously especially is provided.This method can be simultaneously, apace immunoglobulin (Ig) glycosylation, N end pyroglutamic acidization and C end are taken off lysine measures, and can the structure of a small amount of antibody electric charge isomeride be characterized accurately and identify.Particularly, the present invention carries out reduction reaction by the immunoglobulin (Ig) before and after cation-exchange chromatography (CEX-HPLC) analysis protaminase enzyme is cut, its light chain and heavy chain are correctly split, and do not influence original glycosylation of this immunoglobulin (Ig) and end modified situation.Human immunoglobulin(HIg) (being antibody) through reduction carries out the analysis of UHV (ultra-high voltage) liquid chromatograph mass spectrography, can be simultaneously, apace immunoglobulin (Ig) electric charge isomeride (particularly human immunoglobulin(HIg)) glycosylation, N end pyroglutamic acid cyclisation and C end are taken off lysine and measure.
The invention provides a kind of glycosylation of immunoglobulin (Ig) electric charge isomeride and kit of end modified situation measured simultaneously.
Description of drawings
Below, describe embodiment of the present invention in conjunction with the accompanying drawings in detail, wherein:
Figure 1A-1D has shown the comparative result of different DTT consumption antagonist A weight chain separation case among the embodiment 1.
Fig. 2 A-2C has shown that different DTT reduction reaction temperatures among the embodiment 1 separate with time antagonist A weight chain and the influence of end modified situation.
Fig. 3 A-3D has shown the influence comparison of 1,2,3,4 pairs of weight chain chromatographic resolution of gradient situation among the embodiment 1 respectively.
Fig. 4 A-4D has shown different taper hole voltages among the embodiment 1, and (30V is 40V) to the deconvolute influence of chemoattractant molecule amount mass spectra peak intensity of heavy chain for 20V, 25V.
Fig. 5 A has shown the chromatogram that antibody A is measured among the embodiment 2 after reducing; Fig. 5 B1 to Fig. 5 B3 has shown the light chain that antibody A is measured respectively after reducing, pyroglutamic acid light chain and heavy chain mass spectrogram.Fig. 5 C has shown the chromatogram that antibody B measures among the embodiment 2 after reducing; Fig. 5 D1 to Fig. 5 D2 has shown light chain and the heavy chain mass spectrogram that antibody B measures respectively after reducing.PyroE is a N end pyroglutamic acid, and-K is that the C end takes off lysine ,-H
2O is dehydration.
Fig. 6 A shown among the embodiment 3 adopt the protaminase enzyme to cut antibody A after SCX-HPLC analyze and each collected electric charge isomeride; The chromatogram that Fig. 6 B measures after reducing for the electric charge isomeride at peak 1 among Fig. 6 A and peak 4, Fig. 6 C have shown the heavy chain mass spectrogram that the electric charge isomeride at peak 1 and peak 4 is measured among Fig. 6 A after reducing.PyroE is a N end pyroglutamic acid, and-K is that the C end takes off lysine ,-H
2O is dehydration.
Fig. 7 has shown the chromatogram that antibody C (IgG2) measures among the embodiment 4 after reducing.PyroE is a N end pyroglutamic acid, and-K is that the C end takes off lysine ,-H
2O is dehydration.
Embodiment
Followingly the present invention is described with reference to specific embodiment.It will be appreciated by those skilled in the art that these embodiment only are used to illustrate the present invention, the scope that it does not limit the present invention in any way.
Antibody A among the following embodiment is a mosaic type IgG antibody 1, (concrete preparation method is as Chinese patent: shown in the CN 101177453B instructions 10-13 page or leaf embodiment 1-6, wherein the C2-11-12 chimeric antibody that filters out of 13 pages of embodiment 6 of instructions is antibody A of the present invention); Antibody B is a humanization IgG antibody 1, (produce by the beautiful pearl monoclonal antibody in Zhuhai Bioisystech Co., Ltd, concrete preparation method is as Chinese patent: shown in the CN 102675460A instructions 12-18 page or leaf embodiment 1-7, wherein the AT-132 antibody that filters out of instructions 17-18 page or leaf embodiment 7 is antibody B of the present invention); Antibody C is a total man source IgG antibody 2, is produced by AmgenCanada Inc..
Experimental technique among the following embodiment if no special instructions, is conventional method.Used medicinal raw material, reagent material etc. if no special instructions, are commercially available purchase product among the following embodiment.
Embodiment 1The conditional filtering of immunoglobulin (Ig) method of reducing
1.1 investigate the consumption of reductive agent DTT
The DTT that has investigated 4 different amounts is to influence light, that heavy chain separates.Get 4 parts in 5 μ g antibody A albumen, join respectively in the 10 μ L 6M guanidine hydrochloride solutions, add 0.1M DTT solution 2 μ L and 5 μ L more respectively, and 0.5M DTT solution 2 μ L and 4 μ L, adding an amount of 6M guanidine hydrochloride solution at last makes the DTT final concentration be respectively 10mM, 25mM, 50mM and 100mM make itself and described IgG1 albumen react 45min down at 65 ℃.
Adopt the light chain and the heavy chain of the reverse UHV (ultra-high voltage) liquid chromatography of C4 separating reaction gained, the liquid phase systems of using as UPLC (Waters, ACQUITY).Chromatographic column: Waters, ACQUITY UPLC column, BEH C4,1.7 μ m (particle diameter),
(aperture), 2.1 * 50mm.Chromatographic condition is set at: chromatogram column temperature is set at 60 ℃, sample size 1 μ g; Moving phase X is 0.1% formic acid water, and moving phase Y is 0.1% formic acid acetonitrile, and flow velocity is 0.4mL/min; Condition of gradient elution is:
Adopt electrospray ionization mass spectrometry to measure through the light chain of chromatographic resolution acquisition and the molecular weight of heavy chain, wherein at 0-5min, stream leads to waste liquid, 5-16min, and stream leads to mass spectrum, adopts positive ion mode to gather mass spectrometric data then; The mass spectrum condition enactment is: the taper hole air-flow is 50.0L/Hr, and desolventizing gas is 800.0L/Hr, and the desolventizing temperature is 500 ℃, and sweep limit is 400-2500Da, and be 1s sweep time, and sampling taper hole voltage is set at 25V.
The result is shown in Figure 1A-1D, and when the DTT final concentration was 10mM, antibody A weight chain failed to split fully; When DTT concentration is 25M, can splits fully under the most of situation of weight chain, but have utmost point individual samples chromatographic resolution undesirable; When DTT concentration was 50-100mM, all antibody sample weight chains split fully.For guaranteeing that antibody weight chain splits and chromatographic resolution, determine to use the suitable reductive agent consumption of 25-100mM DTT as 0.2-3 μ g/ μ L antibody protein, 50mM is a preferable amount.
1.2 investigate the temperature and time of reduction reaction
Getting 5 μ g antibody A joins respectively in the 10 μ L 6M guanidine hydrochloride solutions for some parts, add 0.5M DTT solution 2 μ L again, adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, investigated that 37 ℃, 50 ℃, 65 ℃ these three temperature of reaction and 20min are light to the reaction time antagonist IgG1 of 120min, heavy chain separates and end modified result's influence.Chromatogram is consistent with embodiment 1.1 with the mass spectrum condition.
Experimental data uses the BiopharmaLynx1.3 software of Waters company to handle.Select " Intact Protein " deconvoluting of pattern (Deconvolution) to handle, method parameter is as follows: Lock Mass (Da): 556.2771; TICThreshold:300-500; Deconvolution m/z Range: light chain is 850-2000, and heavy chain is 950-1500; Protein MW Range: light chain is 20000-30000Da, and heavy chain is 42000-60000Da.The ratio of each sugared type of IgG antibody is carried out normalization according to the intensity of each sugared type molecular weight peaks in the heavy chain mass spectrogram and is calculated.The light chain that contains N end pyroglutamic acid can be realized baseline separation with the light chain that does not contain pyroglutamic acid, therefore chromatographic peak area is carried out integration, can calculate the ratio of light chain N end pyroglutamic acidization.It is to obtain by the molecular weight analyse to the G0F heavy chain that heavy chain N end pyroglutamic acidization and C end take off lysine.
Concrete outcome is shown in Fig. 2 and table 1.Wherein, by Fig. 2 A-1 to Fig. 2 A-3 as seen, when reduction temperature was 37 ℃, antibody weight chain did not separate fully, illustrated that reduction reaction is not thorough under this temperature.By Fig. 2 B-1 to Fig. 2 B-3 and Fig. 2 C-1 to Fig. 2 C-3 as seen, when reduction temperature raise be 50 ℃ and 65 ℃ (reaction time 〉=45min) time, antibody weight chain separation case is more satisfactory, illustrate that the DTT reduction reacts more thorough under the high temperature more than 50 ℃.In addition, as can be seen, under identical reduction temperature, along with the prolongation in reaction time, the modification ratio of sample weight chain end is all in rising trend from following table 1; Equally, in the identical reaction time, along with the rising of temperature of reaction, sample weight chain end is modified ratio also all to be increased gradually.And, consider to promote N end pyroglutamic acidization and C end to take off the lysine reaction under the hot conditions, must investigate the specimen preparation process to end modified influence.As shown in table 1, as 45min≤reaction time≤120min, the amplitude of variation of end modified data is all little, and therefore, the end modified influence of specimen in use preparation condition antagonist is less.
To sum up, determine that reduction reaction conditions is: 50-65 ℃ of reaction down, the reaction time is: 45min≤reaction time≤120min.
1.3 method precision and reappearance
(getting 5 μ g antibody A joins respectively in the 10 μ L 6M guanidine hydrochloride solutions for some parts under the experiment condition after optimization, add 0.5M DTT solution 2 μ L again, adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, 65 ℃ of reaction 45min), estimate this method and measure precision and the reappearance that the glycosylation of antibody A, N end pyroglutamic acid cyclisation and C end take off lysine.Chromatogram is consistent with embodiment 1.1 with the mass spectrum condition.Data processing method is consistent with embodiment 1.2.
METHOD FOR CONTINUOUS DETERMINATION five times, the result is as shown in table 2.The measurement result RSD% that each sugared type ratio of IgG1, N end pyroglutamic acid cyclisation and C end take off the polylysine modification ratio is all less than 0.5%, and sugared type assay result's RSD% is less than 7%.
Measure the sample of five parallel processing, the result is as shown in table 3.The measurement result RSD% that N end pyroglutamic acid cyclisation and C end take off the polylysine modification ratio is all less than 1%, and sugared type assay result's RSD% is less than 6%.
Table 2 method precision is measured
Table 3 method reappearance is measured
To sum up, the precision of this method and reappearance are good.
1.4 moving phase optimization
Based on anti-phase UHV (ultra-high voltage) liquid chromatography, investigated of the influence of moving phase gradient to weight chain chromatographic resolution.Sample uses 5 μ g antibody A to join in the 10 μ L 6M guanidine hydrochloride solutions, adds 0.5M DTT solution 2 μ L again, and adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, 65 ℃ of reaction 45min.Reactor product adopts different eluent gradients to separate, and other chromatographic conditions are consistent with embodiment 1.1 with the mass spectrum condition.Moving phase X is 0.1% formic acid water, and moving phase Y is 0.1% formic acid acetonitrile, and flow velocity is 0.4mL/min, has investigated four kinds of gradient, and is specific as follows:
Gradient 1:0-5min, 10%Y; 5-5.1min, 10%-18%Y; 5.1-15min, 18%-28%Y; 15-15.1min, 28%-90%Y; 15.1-19.0min, 90%Y; 19.0-19.1min, 90%-10%Y, 19.1-22.0min, 10%Y.
Gradient 2:0-5min, 10%Y; 5-5.1min, 10%-25%Y; 5.1-8min, 25%-27%Y; 8-18min, 27%-30%Y; 18-18.1min, 30%-90%Y; 18.1-21.0min, 90%Y; 21.0-21.1min, 90%-10%Y, 21.1-24.0min, 10%Y.
Gradient 3:0-5min, 10%Y; 5-5.1min, 10%-25%Y; 5.1-8min, 25%-26%Y; 8-18min, 26%-28%Y; 18-18.1min, 28%-90%Y; 18.1-21.0min, 90%Y; 21.0-21.1min, 90%-10%Y, 21.1-24.0min, 10%Y.
Gradient 4:0-5min, 10%Y; 5-5.1min, 10%-25%Y; 5.1-6min, 25%-26%Y; 6-10min, 26%-27%Y; 10-15min, 27%-32%Y; 15-15.1min, 32%-90%Y; 15.1-18.0min, 90%Y; 18.0-18.1min, 90%-10%Y, 18.1-21.0min, 10%Y.
The chromatographic resolution result of light chain and heavy chain as shown in Figure 3.To sum up, realize that the optimal flow phase gradient of weight chain baseline separation is gradient 4:0-5min, 10%Y; 5-5.1min, 10%-25%Y; 5.1-6min, 25%-26%Y; 6-10min, 26%-27%Y; 10-15min, 27%-32%Y; 15-15.1min, 32%-90%Y; 15.1-18.0min, 90%Y; 18.0-18.1min, 90%-10%Y, 18.1-21.0min, 10%Y.
1.5 mass spectrum condition optimizing
Based on moving phase gradient (0-5min, the 10%Y after optimizing; 5-5.1min, 10%-25%Y; 5.1-6min, 25%-26%Y; 6-10min, 26%-27%Y; 10-15min, 27%-32%Y; 15-15.1min, 32%-90%Y; 15.1-18.0min, 90%Y; 18.0-18.1min, 90%-10%Y, 18.1-21.0min, 10%Y), investigated the mass spectrum parameter after to baseline separation light chain and the influence of heavy chain mass signal.The taper hole air-flow, desolventizing gas and desolventizing temperature etc. are little to the mass signal influence, adopt instrument supplier proposed parameter substantially.The inventive method is optimized sampling taper hole voltage.Sample uses 5 μ g antibody A to join in the 10 μ L 6M guanidine hydrochloride solutions, adds 0.5M DTT solution 2 μ L again, and adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, 65 ℃ of reaction 45min.Reactor product adopts the chromatographic condition among the embodiment 1.1 to separate, and mass spectrum sampling taper hole voltage is set at 20V respectively, 25V, and 30V and 40V, other mass spectrum conditions are consistent with embodiment 1.1.Shown in Fig. 4 A-1 to Fig. 4 A-4, along with the rising of voltage, the total ion current of weight chain (peak area) all increases to some extent; And shown in Fig. 4 B-1 to Fig. 4 B-4, the chemoattractant molecule amount of the deconvoluting mass signal of heavy chain has between 20-25V the time more obviously to be increased, and does not have significant difference between 25-30V, and remarkable reduction is then arranged when 40V.To sum up, the mass spectrum sampling taper hole voltage of determining this method is 25-30V.
Embodiment 2Adopt UPLC-MS method of the present invention to measure glycosylation and the end modified situation of antibody A and antibody B (IgG1)
(5 μ g antibody A join in the 10 μ L6M guanidine hydrochloride solutions reducing condition after employing is optimized, add 0.5M DTT solution 2 μ L again, adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, 65 ℃ of reaction 45min), UPLC separates (consistent with embodiment 1.1), the glycosylation of ESI-MS detection (consistent with embodiment 1.1) and normalization data processing (with embodiment 1.2 unanimities) analysis antibody A and antibody B and end modified.The N of antibody A light chain and heavy chain holds first amino acid to be glutamine (Gln), and the pyroglutamic acid cyclisation easily takes place; It is glutamic acid (Glu) that antibody B light chain N holds first amino acid, be difficult for the pyroglutamic acid cyclisation takes place, and heavy chain is the glutamine that cyclisation easily takes place.Fig. 5 A is the chromatogram that antibody A adopts UPLC-MS method of the present invention to record through the reduction back, the mass spectrogram that Fig. 5 B records for this antibody, wherein Fig. 5 B-1 is that retention time is the deconvoluting mass spectrogram of literalness light chain (LC) for the 8.19min chromatographic peak among Fig. 5 A, and molecular weight is 23056Da.Fig. 5 B-2 is that retention time is the deconvoluting mass spectrogram of the light chain of N end pyroglutamic acidization for the 9.67min chromatographic peak among Fig. 5 A, and molecular weight is 23039Da.Fig. 5 B-3 is that retention time is the deconvoluting mass spectrogram of heavy chain (HC) for the 11.27min chromatographic peak among Fig. 5 A, the mass spectra peak of different quality number is represented respectively and is contained different sugar type and end modified IgG1 molecular weight among Fig. 5 B-3, and its determining molecular weight and theoretical molecular are as shown in table 4.It is very consistent with its theoretical value that this method records the weight chain molecular weight of antibody A, the accuracy height; And can distinguish the mass spectra peak of the 17Da of being of poor quality, as 50542Da (G0F, pyroglutamic acid take off lysine) and 50559Da (G0F takes off lysine), show the resolution height.The chromatogram that Fig. 5 C adopts UPLC-MS method of the present invention to record for antibody B through the reduction back, the mass spectrogram that Fig. 5 D records for this antibody, wherein Fig. 5 D-1 is that retention time is the deconvoluting mass spectrogram of light chain (LC) for the 11.57min chromatographic peak among Fig. 5 C, and no pyroglutamic acid chemoattractant molecule amount is 23056Da.Fig. 5 D-2 is that retention time is the deconvoluting mass spectrogram of heavy chain (HC) for the 13.26min chromatographic peak among Fig. 5 C, and the mass spectra peak of different quality number is represented respectively and contained different sugar type and end modified IgG1 molecular weight among Fig. 5 D-2.The same with antibody A, the measured value of antibody B weight chain molecular weight is very consistent with theoretical value.Calculate by normalization, antibody B heavy chain N end pyroglutamic acidization and C end take off lysine and are respectively 70.6% and 97.8%; G0F, G1F, the content of G2F and G0 is respectively 65.7%, 26.5%, and 4.6% and 3.2%.
Table 4 antibody A contains different sugar type and end modified heavy chain molecule amount theoretical value and measured value
| Glycosylation and end modified | Theoretical molecular (Da) | Actual measurement molecular weight (Da) |
| G0F, pyroglutamic acid takes off |
50542 | 50542 |
| G0F, pyroglutamic acid takes off lysine, dehydration | 50524 | 50523 |
| G0F takes off lysine | 50559 | 50559 |
| G0F, pyroglutamic acid | 50670 | 50670 |
| G1F, pyroglutamic acid takes off lysine | 50704 | 50704 |
| Man5, pyroglutamic acid takes off lysine | 50314 | 50315 |
| G0F-GN, pyroglutamic acid takes off lysine | 50338 | 50339 |
| G0, pyroglutamic acid takes off lysine | 50395 | 50395 |
Embodiment 3Adopt UPLC-MS method of the present invention to measure the glycosylation and the end modified situation of antibody A electric charge isomeride
Get 200 μ g antibody A, add protaminase 1 μ g respectively, 4 μ g, 10 μ g and 20 μ g, 37 ℃ were reacted about 3 hours.Adopt cation-exchange chromatography (CEX-HPLC) to analyze then; Chromatographic column can adopt Dionnex BioLC MAbPac SCX-104 * 250mm, uses moving phase E (20mM MES and 20mM NaCl) and moving phase F (20mM MES and 200mM NaCl) to carry out gradient elution (0-3min, 10-20%F, 3-25min, 20-50%F, 25-38min, 50-70%F, 38-40min, 70%F, 40-42min, 70-10%F, 42-45min, 10%F).The result shows, enzyme was cut insufficiently when the amount of protaminase was 1 μ g and 4 μ g, and enzyme is cut fully during 〉=10 μ g, therefore preferred 10 μ g protaminases/200 μ g antibody.In addition, get some parts of 200 μ g antibody A, add protaminase 10 μ g, 37 ℃ were reacted 2,3,5 hours, and the result shows that enzyme was cut fully in 〉=3 hours.Therefore, get 200 μ g antibody A, add about 3 hours of 37 ℃ of reactions of protaminase 10 μ g, adopt above-mentioned CEX-HPLC to analyze the antibody (Fig. 6 A) of comparison process front and back then, and collect peak 1, peak 2, peak 3, peak 4 electric charge isomeride in the sample behind the post.
(5 μ g antibody A join in the 10 μ L6M guanidine hydrochloride solutions reducing condition after employing is optimized, add 0.5M DTT solution 2 μ L again, adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, 65 ℃ of reaction 45min), UPLC separates (consistent with embodiment 1.1), ESI-MS detects (consistent with embodiment 1.1) and normalization data and handles (with embodiment 1.2 unanimities) to the glycosylation of IgG1 electric charge isomeride with end modifiedly carry out assay determination, with the textural difference of discriminating electric charge isomeride.The results are shown in Figure 6B-1 to Fig. 6 B-2 and Fig. 6 C-1 to Fig. 6 C-2.
Measurement result shows, the electric charge isomeride takes off at N end pyroglutamic acidization and C end that there were significant differences on the polylysine modification, and to the peak 4, N end pyroglutamic acidization and/or C hold and take off the polylysine modification ratio and reduces gradually by peak 1; Also there is part difference aspect the sugared type content, obviously increasing as the Man5 content of peak 4 than peak 1.Therefore, in conjunction with cation-exchange chromatography, our bright method can be used for the glycosylation of check and analysis antibody electric charge isomeride and end modified difference.
Embodiment 4Adopt UPLC-MS method of the present invention to measure glycosylation and the end modified situation of antibody C (IgG2)
(5 μ g antibody A join in the 10 μ L 6M guanidine hydrochloride solutions reducing condition after optimizing among employing the present invention, add 0.5MDTT solution 2 μ L again, adding an amount of 6M guanidine hydrochloride solution at last, to make the DTT final concentration be 50mM, 65 ℃ of reaction 45min), separate (consistent) through UPLC, the glycosylation of ESI-MS detection (consistent) and normalization data processing (with embodiment 1.2 unanimities) analysis antibody C and end modified with embodiment 1.1 with embodiment 1.1.The chromatogram that Fig. 7 adopts UPLC-MS method of the present invention to record for antibody C through the reduction back, the retention time of light chain (LC) is 6.6min, the retention time of heavy chain (HC) is 13.7min.The N of antibody C light chain and heavy chain holds first amino acid to be glutamic acid (Glu), is difficult for cyclisation and forms pyroglutamic acid, does not therefore detect the light chain or the heavy chain of pyroglutamic acidization; The C end has taken place and has taken off lysine in the heavy chain major part.The sugared type of antibody C mainly comprises G0F, G1F, and Man5, G0 and G2F, the corresponding molecular weight that takes off the lysine heavy chain that contains is 50206Da, 50367Da, 49978Da, 50059Da and 50531Da, consistent with theoretical value; Its content is respectively 58.0%, 19.5%, and 13.7%, 6.6%, 2.2%.Therefore, the inventive method is equally applicable to Immunoglobulin IgG2 glycosylation and end modified detection.
Embodiment 5Adopt the present invention to measure the kit method of the glycosylation and the end modified situation of antibody A
Kit is made of reagent A and reagent B, and wherein reagent A is the 6M guanidine hydrochloride solution; Reagent B is a 0.5M DTT solution.
The glycosylation of use kit detection antibody A and the method for end modified situation are specific as follows:
(protein concentration should be greater than 1 μ g/ μ L to get 20 μ g antibody A, if less than 1 μ g/ μ L, available molecular cut off is that the ultra-filtration centrifuge tube of 10kDa concentrates), adding a certain amount of reagent A to overall solution volume is 36 μ L, add 4 μ L reagent B again, react 45min down at 65 ℃.Reactor product adopts UPLC to separate (consistent with embodiment 1.1), the glycosylation of ESI-MS detection (consistent with embodiment 1.1) and normalization data processing (with embodiment 1.2 unanimities) analysis antibody A and end modified.Carried out repeated experiments (reconfiguring sample and mensuration) in continuous 5 days.
The result shows that the light chain and the heavy chain of antibody A effectively split, and realize baseline separation on chromatogram.As shown in table 5, METHOD FOR CONTINUOUS DETERMINATION 5 days, light chain N holds pyroglutamic acidization, and heavy chain N end pyroglutamic acidization and C end take off the relative standard deviation RSD% of lysine measured value less than 2%; Sugar chain G0F, the relative standard deviation RSD% of G1F and G0 relative content measured value is less than 5%, and Man5 and G0F-GN are less than 10%.To sum up, this method can realize normalizing operation, and reappearance is good, can be used for setting up mensuration immunoglobulin (Ig) glycosylation and end modified kit method.
Table 5 is used the reappearance that this method is measured the end modified situation of antibody A
More than specific description of embodiments of the present invention does not limit the present invention, those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (10)
1. measure the glycosylation of immunoglobulin (Ig) electric charge isomeride and the method for end modified situation for one kind, said method comprising the steps of:
1) use cation-exchange chromatography (CEX-HPLC) to analyze the immunoglobulin (Ig) that the protaminase enzyme is cut front and back, and by collecting different immunoglobulin (Ig) electric charge isomeride behind the retention time post;
2) make in the step 1) immunoglobulin (Ig) after the denaturant sex change, use the reductive agent reduction, thereby split light chain and heavy chain;
3) light chain and the heavy chain of immunoglobulin (Ig) the anti-phase UHV (ultra-high voltage) liquid chromatography separating step 2 of use);
4) light chain that obtains in the use mass spectroscopy step 3) and the molecular weight of heavy chain; And
5) analytical procedure 3) in chromatographic data and the mass spectrometric data in the step 4), thereby measure the glycosylation and the end modified situation of described immunoglobulin (Ig).
2. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 1 and the method for end modified situation is characterized in that the mass ratio of described protaminase and immunoglobulin (Ig) is 1: 20; The enzyme time of cutting is 3 hours.
3. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 1 and the method for end modified situation, it is characterized in that, described step 2) comprising: the 1-6M guanidine hydrochloride aqueous solution that in a certain amount of immunoglobulin (Ig), adds 10-30 μ L, mix the back and add 1-4 μ L dithiothreitol (DTT) (DTT) aqueous solution, make immunoglobulin (Ig) generation sex change, reduction reaction, wherein, the DTT final concentration is 25-100mM in the reaction solution, and the immunoglobulin (Ig) final concentration is 0.2-3 μ g/ μ L.
4. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 3 and the method for end modified situation is characterized in that described step 2) in the DTT final concentration be 50mM.
5. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 3 and the method for end modified situation, it is characterized in that, described step 2) immunoglobulin (Ig) generation sex change in, reduction reaction temperature are 50-65 ℃, and the reaction time is 45min-120min.
6. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 5 and the method for end modified situation is characterized in that described step 2) in immunoglobulin (Ig) generation sex change, reduction reaction temperature be 65 ℃, the reaction time is 45min.
7. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 1 and the method for end modified situation, it is characterized in that, described step 3) comprises: the light chain and the heavy chain of immunoglobulin (Ig) the reverse UHV (ultra-high voltage) liquid chromatography of employing C4 separating step 2), and to realize the baseline separation of described light chain and heavy chain.
8. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 1 and the method for end modified situation is characterized in that described step 4) comprises:
Employing electrospray ionization mass spectrometry determination step 3) light chain that obtains in and the molecular weight of heavy chain, wherein at 0-5min, stream leads to waste liquid, 5-16min, stream leads to mass spectrum, adopts positive ion mode collection mass spectrometric data then.
The mass spectrum condition enactment is:
The taper hole air-flow is 50.0L/Hr, and desolventizing gas is 800.0L/Hr, and the desolventizing temperature is 500 ℃, and taper hole voltage is 20-40V, and sweep limit is 400-2500Da, and be 1s sweep time.
9. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 1 and the method for end modified situation is characterized in that described step 5) comprises:
Calculated the N end pyroglutamic acid ratio of the light chain of described immunoglobulin (Ig) by the chromatographic peak area that obtains in the step 3), the sugared type relative content and the N that are calculated the heavy chain of described immunoglobulin (Ig) by the mass spectrometric data that obtains in the step 4) hold pyroglutamic acidization and C end to take off lysine ratio.
10. the glycosylation of mensuration immunoglobulin (Ig) electric charge isomeride according to claim 1 and the method for end modified situation is characterized in that described immunoglobulin (Ig) is a human immunoglobulin(HIg).
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| CN111758029A (en) * | 2018-02-27 | 2020-10-09 | 新加坡科技研究局 | Methods, apparatus and computer readable media for glycopeptide identification |
| CN111758029B (en) * | 2018-02-27 | 2023-06-09 | 新加坡科技研究局 | Methods, apparatus, and computer readable media for glycopeptide identification |
| CN111458396A (en) * | 2019-01-18 | 2020-07-28 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
| CN111458396B (en) * | 2019-01-18 | 2022-07-08 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
| CN109932444A (en) * | 2019-03-19 | 2019-06-25 | 北京泰德制药股份有限公司 | A kind of evaluation method of a variety of charge isomer posttranslational modifications of glycoprotein |
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Denomination of invention: A method for determining the glycosylation and end modification of immunoglobulin charge isomers Effective date of registration: 20231215 Granted publication date: 20151202 Pledgee: CITIC Bank Zhuhai Branch Pledgor: LIVZON MABPHARM Inc. Registration number: Y2023980072292 |