CN103217499B - A kind ofly measure the glycosylation of immunoglobulin charge isomer and the method for end modified situation - Google Patents
A kind ofly measure the glycosylation of immunoglobulin charge isomer and the method for end modified situation Download PDFInfo
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Abstract
The invention provides and a kind ofly measure the glycosylation of immunoglobulin charge isomer and the method for end modified situation, the method can hold pyroglutamic acid and the de-lysine of C end to measure to immunoglobulin (Ig) glycosylation, N simultaneously, rapidly, said method comprising the steps of: 1) immunoglobulin (Ig) that protaminase enzyme cuts front and back is analyzed in use cation-exchange chromatography (CEX-HPLC), and collects different immunoglobulin charge isomer by after retention time post; 2) step 1 is made) middle immunoglobulin (Ig) is after denaturant sex change, and use reductive agent reduces, thus splits light chain and heavy chain; 3) use anti-phase ultrahigh pressure liquid phase chromatrographic separation step 2) in the light chain of immunoglobulin (Ig) and heavy chain; 4) use mass spectroscopy step 3) in the molecular weight of the light chain that obtains and heavy chain; And 5) analytical procedure 3) in chromatographic data and step 4) in mass spectrometric data, thus measure the glycosylation of described immunoglobulin (Ig) and end modified situation.
Description
Technical field
The present invention relates to biological technical field.Specifically, the invention provides a kind of glycosylation of Simultaneously test immunoglobulin charge isomer and the method for end modified situation, the invention still further relates to simultaneously and a kind ofly measure the glycosylation of immunoglobulin charge isomer and the kit of end modified situation.
Background technology
Nearly ten years, monoclonal antibody obtains great success and huge development in biological medicine circle and even whole pharmaceuticals industry.The advantages such as compared with traditional small-molecule drug, monoclonal antibody has high specificity, evident in efficacy, and spinoff is little, and consumption is few.With regard to drug molecule characteristic, antibody has larger inhomogeneity.This characteristic of antibody is caused by many factors, and posttranslational modification is wherein most important internal factor.Modify after common antibody translation and comprise glycosylation, N holds pyroglutamic acid, the de-lysine of C end, deacylated tRNA amine, oxidation, isomerization etc.In multiple links of antibody drug research and development, as Molecular Identification, process exploitation, quality monitoring etc., all need that detection is carried out to posttranslational modification and analyze.
IgG antibody glycosylation occurs in the asparagine in heavy chain Fc district, belongs to N-glycosylation, is the important feature composition of antibody.The core cell of IgG sugar chain is made up of two fork anatomical connectivity of two N-Acetyl-D-glucosamines 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 sugar-type and content.Glycosylation difference can affect the biologic activity of antibody and medicine for feature, as removed the half life period etc. in CDC, ADCC, body.
When the N terminal amino acid of IgG antibody is glutamine, easily there is cyclization and generate pyroglutamic acid (pyroglutamicacid, pyroE).This reaction can spontaneously be carried out, and also can enzymatically carry out.Hold at the C of IgG antibody molecule, then de-lysine (-K) easily occurs.In most cases, the biologically active of both antagonists does not affect, but the N of some antibody holds pyroglutamic acidization may affect the adhesion of itself and antigen also to have report to refer to.In addition, the glutamine pyroglutamic acid of N end and the de-lysine of C end all can affect the CHARGE DISTRIBUTION of antibody, and charge characteristic is one of important indicator of antibody mass monitoring.
Therefore, set up and detect fast IgG antibody glycosylation and end modified analytical approach is significant in antibody research and development.At present, this area is generally to glycosylation with end modifiedly to detect separately.Enzymolysis fluorescent marker method is the classical quantivative approach that IgG1 glycosylation measures, but sample handling processes is quite complicated, length consuming time, and needs sample size large; Also have and use mass spectroscopy IgG enzymatic fragment to carry out glycosylation analysis, as papain enzyme and IdeS enzyme etc., but these methods all come with some shortcomings, as restriction enzyme site selectivity is not strong, or high cost, or sample preparation complexity etc., be not suitable for the batch detection of routine or process exploitation sample.Application LC-MS carries out peptide figure analysis can detect the glycosylation of antibody and end modified in theory simultaneously, but there are many technological difficulties in the separation of sugary polypeptide, mensuration and data analysis, be not suitable for glycosylation quantitative test, and sample handling processes is complicated, length consuming time, enzyme cuts through journey also may antagonist be original end modifiedly has an impact.Therefore, do not have the glycosylation to IgG and the end modified report simultaneously carrying out Fast Measurement that are applicable to antibody research and development at present yet, do not have yet can carry out fast the structure of a small amount of antibody charge isomer, the method for accurate characterization and qualification.
Summary of the invention
In order to solve the problems of the technologies described above, the object of this invention is to provide a kind of glycosylation of Simultaneously test immunoglobulin (Ig) (i.e. antibody) charge isomer and the method for end modified situation, the method can hold pyroglutamic acid and the de-lysine of C end to measure to immunoglobulin (Ig) glycosylation, N simultaneously, rapidly, can characterize accurately the structure of a small amount of antibody charge isomer and identify.Simultaneously another object of the present invention is to provide and a kind ofly measures the glycosylation of immunoglobulin charge isomer and the kit of end modified situation.
The invention provides and a kind ofly measure the glycosylation of immunoglobulin charge isomer and the method for end modified situation, said method comprising the steps of:
1) immunoglobulin (Ig) that protaminase enzyme cuts front and back is analyzed in use cation-exchange chromatography (CEX-HPLC), and collects different immunoglobulin charge isomer by after retention time post;
2) step 1 is made) middle immunoglobulin (Ig) is after denaturant sex change, and use reductive agent reduces, thus splits light chain and heavy chain;
3) use anti-phase ultrahigh pressure liquid phase chromatrographic separation step 2) in the light chain of immunoglobulin (Ig) and heavy chain;
4) use mass spectroscopy step 3) in the molecular weight of the light chain that obtains and heavy chain; And
5) analytical procedure 3) in chromatographic data and step 4) in mass spectrometric data, thus measure the glycosylation of described immunoglobulin (Ig) and end modified situation.
Wherein, described immunoglobulin (Ig) is preferably human immunoglobulin(HIg), is preferably human immunoglobulin(HIg) IgG, more preferably human immunoglobulin(HIg) IgG1 and IgG2 hypotype.
Further, the light chain N that the glycosylation of described immunoglobulin (Ig) and end modified situation preferably include immunoglobulin (Ig) holds pyroglutamic acid, and the Asn glycosylation of heavy chain and N hold the de-lysine of pyroglutamic acid, C end.
In assay method provided by the invention, described step 1) in protaminase and the mass ratio of immunoglobulin (Ig) be 1: 20; The enzyme time of cutting is 3 hours.
Particularly, described step 2) comprising: the 1-6M aqueous guanidinium-H CL adding 10-30 μ L in a certain amount of immunoglobulin (Ig), 1-4 μ L dithiothreitol (DTT) (DTT) aqueous solution is added after mixing, make immunoglobulin (Ig) generation sex change, reduction reaction, wherein, in reaction solution, DTT final concentration is 25-100mM, and immunoglobulin (Ig) final concentration is 0.2-3 μ g/ μ L.
Preferably, described step 2) in DTT final concentration be 50mM.
Preferably, described step 2) in immunoglobulin (Ig) generation sex change, reduction reaction temperature be 50-65 DEG C, the reaction time is 45min-120min.
More preferably, described step 2) in immunoglobulin (Ig) generation sex change, reduction reaction temperature be 65 DEG C, the reaction time is 45min.
Particularly, described step 3) specifically comprise:
Adopt reverse ultrahigh pressure liquid phase chromatrographic separation step 2) in the light chain of immunoglobulin (Ig) and heavy chain, to realize the baseline separation of described light chain and heavy chain, according to specific embodiment of the invention scheme, the liquid phase systems of employing can be UPLC (Waters, ACQUITY).Chromatographic column: Waters, ACQUITYUPLCcolumn, BEHC4,1.7 μm (particle diameter),
(aperture), 2.1 × 50mm.
Mobile phase elution requirement affects comparatively greatly with being separated of heavy chain light chain, and preferably, chromatographic condition is set as:
Chromatogram column temperature is set as 55-65 DEG C, sample size 0.2-3 μ g;
Mobile phase X is 0.1% formic acid water, and mobile 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 comprise:
Adopt electrospray ionization mass spectrometry determination step 3) in the molecular weight of the light chain that obtains and heavy chain, wherein at 0-5min, stream leads to waste liquid, 5-16min, and stream leads to mass spectrum, then adopts positive ion mode to gather mass spectrometric data;
Preferably, Mass Spectrometry Conditions is set as:
Taper hole air-flow is 50.0L/Hr, and desolvation gas is 500-800.0L/Hr, and desolventizing temperature is 350-500 DEG C, and sweep limit is 400-2500Da, and sweep time is 0.5-2s.Sampling taper hole voltage is comparatively large on mass signal impact, is set as 20-40V, preferably, is set as 20-30V.
Particularly, described step 5) specifically comprise:
By step 3) in the chromatographic peak area that the obtains N that calculates the light chain of described immunoglobulin (Ig) hold pyroglutamic acid ratio, by step 4) the middle mass spectrometric data obtained is by deconvoluting calculating, and the sugar-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.
The present invention by cation-exchange chromatography (CEX-HPLC) analyze protaminase enzyme cut before and after immunoglobulin (Ig) carry out reduction reaction time, the disulfide bond of albumen ruptures under the effect of DTT, generates the heavy chain that two light chains are equally the same with two.Use C4 anti-phase-ultrahigh pressure liquid phase chromatographic resolution weight chain potpourri, containing or do not hold light chain and the heavy chain of pyroglutamic acid (containing different sugar-type and end modified) can baseline separation be realized, then its molecular weight of employing ESI-MS on-line determination containing N.Owing to having different molecular weight containing different sugar-type or end modified heavy chain, and the ratio of various heavy chain is directly proportional to the intensity of its molecular weight peaks, therefore sugar-type relative content and heavy chain N hold the de-lysine ratio of pyroglutamic acid, C end all can calculate acquisition by heavy chain mass spectrometric data.In addition, light chain N holds pyroglutamic acid ratio to calculate acquisition by chromatographic peak area.
Current this area has some technology to measure molecular weight or its modification situation of protein, but compared with method of the present invention, all existing defects or weak points.Such as mass spectrometer (MS), as MALDI-MS, is usually used in the molecular weight measuring antibody intact proteins level.Although mass spectrometry method is good to sample compatibility, easy to operate, record molecular weight results resolution lower; As the glycosylated classical quantivative approach of IgG antibody---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; The method selectivity is strong, and accuracy is high, but sample handling processes is complicated, length consuming time (usually needing 2 days), and needs sample size comparatively large (being generally at least 100 μ g); Also have and use ESI-MS mensuration IgG enzymatic fragment to carry out glycosylation analysis, but the selectivity of papain restriction enzyme site is low, can increase accessory substance and affect data analysis; Immunoglobulin G digestive enzyme S (IdeS) selectivity is very strong, but the cost of IdeS is high, is not suitable for the batch detection of routine or process exploitation sample; Application LC-MS carries out tryptic mapping analysis theories can detect simultaneously the glycosylation of antibody and end modified, but there are many technological difficulties in the separation of sugary polypeptide, mensuration and data analysis, and its detection sensitivity is low, be unsuitable for the detection of low content glycosyl, in addition sample handling processes is complicated, length consuming time, enzyme cuts through that Cheng Keneng antagonist is original end modifiedly to have an impact.
By contrast, this method adopts ESI-MS to measure antibody multiple-charged ion, then carries out deconvoluting and calculates, substantially increase the accuracy (< 30ppm) of resolution and testing result.In addition, inventive samples process is simple, reductive agent is only needed to carry out reacting (45min), amount of samples few (5 μ g), and the UPLC-MS detection of sample only need complete in 16min, just can obtain antibody glycosylation, N holds pyroglutamic acid and the data of the de-lysine of C end simultaneously.The present invention is particularly useful for the few experiment of sample size, and as colony screening, and the batch of process exploitation process detects fast; Meanwhile, the present invention is also applicable to the sample detection of conventional amount used (100 μ g).Adopt method of the present invention, can also the structure of antagonist charge isomer carry out characterizing and identifying.
Immunoglobulin (Ig) can be divided into IgG, IgA, IgM, IgD, IgE five class, and wherein IgG can be divided into the hypotypes such as IgG1, IgG2, IgG3, IgG4, and the monoclonal antibody drug 70%-80% sold in the market belongs to IgG1 albuminoid.According to the composition of people source amino acid sequence in IgG antibody 1, the multiple protein such as Chimeric antibodies IgG1, humanized antibody IgG1 can be divided into again.The invention provides a kind of glycosylation of Simultaneously test immunoglobulin charge isomer and the method for end modified situation, the glycosylation of a kind of Simultaneously test Immunoglobulin IgG1, IgG2 and the method for end modified situation are especially provided.The method can hold pyroglutamic acid and the de-lysine of C end to measure to immunoglobulin (Ig) glycosylation, N simultaneously, rapidly, can characterize accurately the structure of a small amount of antibody charge isomer and identify.Specifically, the present invention cuts front and back immunoglobulin (Ig) by analyzing protaminase enzyme to cation-exchange chromatography (CEX-HPLC) carries out reduction reaction, its light chain and heavy chain can be made correctly to split, and do not affect the original glycosylation of this immunoglobulin (Ig) and end modified situation.Human immunoglobulin(HIg) (i.e. antibody) through reduction carries out the analysis of ultrahigh pressure liquid phase chromatograph-mass spectrometer coupling, can pyroglutamic acid cyclisation and the de-lysine of C end be held to measure to immunoglobulin charge isomer (particularly human immunoglobulin(HIg)) glycosylation, N simultaneously, rapidly.
The invention provides simultaneously and a kind ofly measure the glycosylation of immunoglobulin charge isomer and the kit of end modified situation.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Figure 1A-1D shows the comparative result of different DTT consumption antagonist A weight chain separation case in embodiment 1.
Fig. 2 A-2C shows different DTT reduction reaction temperature in embodiment 1 and is separated the impact with end modified situation with time antagonist A weight chain.
Fig. 3 A-3D respectively illustrates the impact of gradient 1,2,3,4 on weight chain chromatographic resolution situation in embodiment 1 and compares.
Fig. 4 A-4D shows different taper hole voltage (20V, 25V, 30V, 40V) in embodiment 1 and to deconvolute on heavy chain the impact of chemoattractant molecule amount mass spectra peak intensity.
Fig. 5 A shows the chromatogram that in embodiment 2, antibody A measures after reduction; Fig. 5 B1 to Fig. 5 B3 respectively illustrates light chain, pyroglutamic acid light chain and the heavy chain mass spectrogram that antibody A measures after reduction.Fig. 5 C shows the chromatogram that in embodiment 2, antibody B measures after reduction; Fig. 5 D1 to Fig. 5 D2 respectively illustrates light chain and the heavy chain mass spectrogram of antibody B mensuration after reduction.PyroE is that N holds pyroglutamic acid, and-K is the de-lysine of C end ,-H
2o is dehydration.
After Fig. 6 A shows in embodiment 3 and adopts protaminase enzyme to cut antibody A, SCX-HPLC analyzes and collected each charge isomer; Fig. 6 B is the chromatogram that the charge isomer at peak 1 and peak 4 in Fig. 6 A measures after reduction, the heavy chain mass spectrogram that the charge isomer that Fig. 6 C shows peak 1 and peak 4 in Fig. 6 A measures after reduction.PyroE is that N holds pyroglutamic acid, and-K is the de-lysine of C end ,-H
2o is dehydration.
Fig. 7 shows the chromatogram that in embodiment 4, antibody C (IgG2) measures after reduction.PyroE is that N holds pyroglutamic acid, and-K is the de-lysine of C end ,-H
2o is dehydration.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiments are only for illustration of the present invention, its scope do not limited the present invention in any way.
Antibody A in following embodiment is Chimeric antibodies IgG1, (concrete preparation method is as Chinese patent: shown in CN101177453B instructions 10-13 page embodiment 1-6, and the C2-11-12 chimeric antibody that wherein instructions 13 pages of embodiments 6 filter out is antibody A of the present invention); Antibody B is humanization IgG antibody 1, (produced by Zhuhai Li Zhu monoclonal antibody Bioisystech Co., Ltd, concrete preparation method is as Chinese patent: shown in CN102675460A instructions 12-18 page embodiment 1-7, the AT-132 antibody that wherein instructions 17-18 page embodiment 7 filters out is antibody B of the present invention); Antibody C is human antibody IgG2, is produced by AmgenCanadaInc..
Experimental technique in following embodiment, if no special instructions, is conventional method.Medicinal raw material used in following embodiment, reagent material etc., if no special instructions, be commercially available purchase product.
embodiment 1the conditional filtering of immunoglobulin (Ig) method of reducing
The consumption of 1.1 investigation reductive agent DTT
The impact that the DTT having investigated 4 different amounts is separated light, heavy chain.Get 4 parts, 5 μ g antibody A albumen, join in 10 μ L6M guanidine hydrochloride solutions respectively, add 0.1MDTT solution 2 μ L and 5 μ L more respectively, and 0.5MDTT solution 2 μ L and 4 μ L, finally adding appropriate 6M guanidine hydrochloride solution makes DTT final concentration be respectively 10mM, 25mM, 50mM and 100mM, make itself and described IgG1 albumen react 45min at 65 DEG C.
Adopt light chain and the heavy chain of C4 reverse ultrahigh pressure liquid phase chromatographic resolution reaction gained, the liquid phase systems of use is UPLC (Waters, ACQUITY).Chromatographic column: Waters, ACQUITYUPLCcolumn, BEHC4,1.7 μm (particle diameter),
(aperture), 2.1 × 50mm.Chromatographic condition is set as: chromatogram column temperature is set as 60 DEG C, sample size 1 μ g; Mobile phase X is 0.1% formic acid water, and mobile 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 the molecular weight of light chain and the heavy chain obtained through chromatographic resolution, wherein at 0-5min, stream leads to waste liquid, 5-16min, and stream leads to mass spectrum, then adopts positive ion mode to gather mass spectrometric data; Mass Spectrometry Conditions is set as: taper hole air-flow is 50.0L/Hr, and desolvation gas is 800.0L/Hr, and desolventizing temperature is 500 DEG C, and sweep limit is 400-2500Da, and sweep time is 1s, and sampling taper hole voltage sets is 25V.
Result as shown in figures 1 a-1d, when DTT final concentration is 10mM, fail to split completely by antibody A weight chain; When DTT concentration is 25M, weight chain can split completely in most cases, but has pole individual samples chromatographic resolution undesirable; When DTT concentration is 50-100mM, all antibody samples weight chains split completely.For ensureing that antibody weight chain splits and chromatographic resolution, determine the Suitable reducing agent consumption using 25-100mMDTT as 0.2-3 μ g/ μ L antibody protein, 50mM is preferable amount.
The temperature and time of 1.2 investigation reduction reactions
Getting the some parts of 5 μ g antibody A joins in 10 μ L6M guanidine hydrochloride solutions respectively, add 0.5MDTT solution 2 μ L again, finally adding appropriate 6M guanidine hydrochloride solution makes DTT final concentration be 50mM, and the reaction time antagonist IgG1 having investigated 37 DEG C, 50 DEG C, 65 DEG C these three temperature of reaction and 20min to 120min is light, heavy chain is separated and the impact of end modified result.Chromatogram is consistent with embodiment 1.1 with Mass Spectrometry Conditions.
Experimental data uses the BiopharmaLynx1.3 software of Waters company to process." IntactProtein " pattern of selection carries out deconvoluting (Deconvolution) process, and method parameter is as follows: LockMass (Da): 556.2771; TICThreshold:300-500; Deconvolutionm/zRange: light chain is 850-2000, heavy chain is 950-1500; ProteinMWRange: light chain is 20000-30000Da, heavy chain is 42000-60000Da.The ratio of each sugar-type of IgG antibody is normalized according to the intensity of sugar-type molecular weight peaks each in heavy chain mass spectrogram and calculates.Hold the light chain of pyroglutamic acid can realize baseline separation with the light chain not containing pyroglutamic acid containing N, therefore integration is carried out to chromatographic peak area, the ratio that light chain N holds pyroglutamic acid can be calculated.Heavy chain N holds pyroglutamic acid and the de-lysine of C end to be by obtaining the molecular weight analyse of G0F heavy chain.
Concrete outcome as shown in Figure 2 and Table 1.Wherein, from Fig. 2 A-1 to Fig. 2 A-3, when reduction temperature is 37 DEG C, antibody weight chain does not separate completely, and under this temperature is described, reduction reaction is not thorough.From Fig. 2 B-1 to Fig. 2 B-3 and Fig. 2 C-1 to Fig. 2 C-3, when reduction temperature raise be 50 DEG C and 65 DEG C (reaction time >=45min) time, antibody weight chain separation case is more satisfactory, reacts more thorough under the high temperature of DTT reduction more than 50 DEG C is described.In addition, as can be seen from following table 1, under identical reduction temperature, along with the prolongation in reaction time, the modification ratio of sample weight chain end is all in rising trend; Equally, in the identical reaction time, along with the rising of temperature of reaction, sample weight chain end modifies ratio also to be increased all gradually.Further, may promote under considering hot conditions that N holds pyroglutamic acid and the de-lysine reaction of C end, Sample Preparation Procedure must be investigated on end modified impact.As shown in table 1, when 45min≤reaction time≤120min, the amplitude of variation of end modified data is all little, and therefore, the end modified impact of specimen in use preparation condition antagonist is less.
Under table 1 different DTT reduction temperature and time conditions, the end modified situation of IgG antibody 1 compares
To sum up, determine that reduction reaction conditions is: react at 50-65 DEG C, the reaction time is: 45min≤reaction time≤120min.
1.3 method precisions and reappearance
Under experiment condition after optimization, (getting the some parts of 5 μ g antibody A joins in 10 μ L6M guanidine hydrochloride solutions respectively, add 0.5MDTT solution 2 μ L again, finally adding appropriate 6M guanidine hydrochloride solution makes DTT final concentration be 50mM, 65 DEG C of reaction 45min), evaluate precision and reappearance that the glycosylation of this method mensuration antibody A, N end pyroglutamic acid cyclisation and C hold de-lysine.Chromatogram is consistent with embodiment 1.1 with Mass Spectrometry Conditions.Data processing method is consistent with embodiment 1.2.
METHOD FOR CONTINUOUS DETERMINATION five times, result is as shown in table 2.The each sugar-type ratio of IgG1, N hold the measurement result RSD% of pyroglutamic acid cyclisation and the de-polylysine modification ratio of C end to be all less than 0.5%, and the RSD% of sugar-type assay result is less than 7%.
Measure the sample of five parallel processing, result is as shown in table 3.N holds the measurement result RSD% of pyroglutamic acid cyclisation and the de-polylysine modification ratio of C end to be all less than 1%, and the RSD% of sugar-type assay result is less than 6%.
Table 2 method precision measures
Table 3 method reappearance measures
To sum up, the precision of the method and reappearance good.
1.4 Optimization of mobile phase
Based on anti-phase ultrahigh pressure liquid phase chromatogram, investigate the impact of mobile phase gradient on weight chain chromatographic resolution.Sample uses 5 μ g antibody A to join in 10 μ L6M guanidine hydrochloride solutions, then adds 0.5MDTT solution 2 μ L, finally adds appropriate 6M guanidine hydrochloride solution and makes DTT final concentration be 50mM, 65 DEG C of reaction 45min.Reactor product adopts different eluent gradient to be separated, and other chromatographic conditions are consistent with embodiment 1.1 with Mass Spectrometry Conditions.Mobile phase X is 0.1% formic acid water, and mobile phase Y is 0.1% formic acid acetonitrile, and flow velocity is 0.4mL/min, has investigated four kinds of gradient, 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, the optimal flow phase gradient realizing 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 Spectrometry Conditions optimizations
Based on mobile phase gradient (0-5min, 10%Y after optimization; 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), investigate the impact of mass spectrometry parameters on the light chain after baseline separation and heavy chain mass signal.Taper hole air-flow, desolvation gas and desolventizing temperature etc. are little on mass signal impact, basic employing instrument supplier proposed parameter.The inventive method is optimized sampling taper hole voltage.Sample uses 5 μ g antibody A to join in 10 μ L6M guanidine hydrochloride solutions, then adds 0.5MDTT solution 2 μ L, finally adds appropriate 6M guanidine hydrochloride solution and makes DTT final concentration be 50mM, 65 DEG C of reaction 45min.Reactor product adopts the chromatographic condition in embodiment 1.1 to be separated, and MS acquisition taper hole voltage is set as 20V, 25V, 30V and 40V respectively, and other Mass Spectrometry Conditions are consistent with embodiment 1.1.As shown in Fig. 4 A-1 to Fig. 4 A-4, along with the rising of voltage, the total ion current (peak area) of weight chain increases all to some extent; And as shown in Fig. 4 B-1 to Fig. 4 B-4, having when chemoattractant molecule amount of the deconvoluting mass signal of heavy chain is between 20-25V more obviously increases, between 25-30V, there is no significant difference, then have remarkable reduction when 40V.To sum up, the MS acquisition taper hole voltage determining the method is 25-30V.
embodiment 2uPLC-MS method of the present invention is adopted to measure the glycosylation of antibody A and antibody B (IgG1) and end modified situation
(5 μ g antibody A join in 10 μ L6M guanidine hydrochloride solutions to adopt the reducing condition after optimizing, add 0.5MDTT solution 2 μ L again, finally adding appropriate 6M guanidine hydrochloride solution makes DTT final concentration be 50mM, 65 DEG C of reaction 45min), UPLC is separated (consistent with embodiment 1.1), and ESI-MS detects (consistent with embodiment 1.1) and normalization data process (consistent with embodiment 1.2) and analyzes the glycosylation of 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), easily pyroglutamic acid cyclisation occurs; Antibody B light chain N holds first amino acid to be glutamic acid (Glu), not easily pyroglutamic acid cyclisation occurs, and heavy chain is the glutamine that cyclisation easily occurs.Fig. 5 A is the chromatogram that antibody A adopts UPLC-MS method of the present invention to record after reduction, Fig. 5 B is the mass spectrogram that this antibody records, wherein Fig. 5 B-1 is that in Fig. 5 A, retention time is the deconvoluting mass spectrogram of 8.19min chromatographic peak and literalness light chain (LC), and molecular weight is 23056Da.Fig. 5 B-2 is that in Fig. 5 A, retention time is the deconvoluting mass spectrogram that 9.67min chromatographic peak and N hold the light chain of pyroglutamic acid, and molecular weight is 23039Da.Fig. 5 B-3 is that in Fig. 5 A, retention time is the deconvoluting mass spectrogram of 11.27min chromatographic peak and heavy chain (HC), in Fig. 5 B-3, the mass spectra peak of different quality number represents containing different sugar-type and end modified IgG1 molecular weight respectively, its determining molecular weight and theoretical molecular as shown in table 4.The weight chain molecular weight that the method records antibody A is very consistent with its theoretical value, and accuracy is high; And can distinguish of poor quality to be the mass spectra peak of 17Da, as 50542Da (G0F, pyroglutamic acid, de-lysine) and 50559Da (G0F takes off lysine), to show that resolution is high.Fig. 5 C is the chromatogram that antibody B adopts UPLC-MS method of the present invention to record after reduction, Fig. 5 D is the mass spectrogram that this antibody records, wherein Fig. 5 D-1 is that in Fig. 5 C, retention time is the deconvoluting mass spectrogram of 11.57min chromatographic peak and light chain (LC), is 23056Da without pyroglutamic acid chemoattractant molecule amount.Fig. 5 D-2 is that in Fig. 5 C, retention time is the deconvoluting mass spectrogram of 13.26min chromatographic peak and heavy chain (HC), and in Fig. 5 D-2, the mass spectra peak of different quality number represents respectively containing different sugar-type and end modified IgG1 molecular weight.The same with antibody A, the measured value of antibody B weight chain molecular weight is very consistent with theoretical value.Calculated by normalization, antibody B heavy chain N holds pyroglutamic acid and the de-lysine of C end to be respectively 70.6% and 97.8%; The content of G0F, G1F, G2F and G0 is respectively 65.7%, 26.5%, 4.6% and 3.2%.
Table 4 antibody A is containing 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, de-lysine | 50542 | 50542 |
| G0F, pyroglutamic acid, de-lysine, dehydration | 50524 | 50523 |
| G0F, de-lysine | 50559 | 50559 |
| G0F, pyroglutamic acid | 50670 | 50670 |
| G1F, pyroglutamic acid, de-lysine | 50704 | 50704 |
| Man5, pyroglutamic acid, de-lysine | 50314 | 50315 |
| G0F-GN, pyroglutamic acid, de-lysine | 50338 | 50339 |
| G0, pyroglutamic acid, de-lysine | 50395 | 50395 |
embodiment 3uPLC-MS method of the present invention is adopted to measure the glycosylation of antibody A charge isomer and end modified situation
Get 200 μ g antibody A, add protaminase 1 μ g respectively, 4 μ g, 10 μ g and 20 μ g, 37 DEG C are reacted about 3 hours.Then cation-exchange chromatography (CEX-HPLC) is adopted to analyze; Chromatographic column can adopt DionnexBioLCMAbPacSCX-104 × 250mm, uses mobile phase E (20mMMES and 20mMNaCl) and mobile phase F (20mMMES and 200mMNaCl) 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).Result shows, when the amount of protaminase is 1 μ g and 4 μ g, enzyme is cut insufficient, and during >=10 μ g, enzyme is cut fully, therefore preferred 10 μ g protaminase/200 μ g antibody.In addition, get the some parts of 200 μ g antibody A, add protaminase 10 μ g, 37 DEG C of reactions 2,3,5 hours, result showed, and within >=3 hours, enzyme is cut fully.Therefore, get 200 μ g antibody A, add protaminase 10 μ g37 DEG C reaction about 3 hours, then adopt the antibody (Fig. 6 A) before and after the process of above-mentioned CEX-HPLC com-parison and analysis, and after post, collect peak 1 in sample, peak 2, peak 3, peak 4 charge isomer.
(5 μ g antibody A join in 10 μ L6M guanidine hydrochloride solutions to adopt the reducing condition after optimizing, add 0.5MDTT solution 2 μ L again, finally adding appropriate 6M guanidine hydrochloride solution makes DTT final concentration be 50mM, 65 DEG C of reaction 45min), UPLC is separated (consistent with embodiment 1.1), ESI-MS detects (consistent with embodiment 1.1) and normalization data process (consistent with embodiment 1.2) to the glycosylation of IgG1 charge isomer with end modifiedly carry out analysis and measure, to differentiate the textural difference of charge isomer.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 display, there were significant differences on N end pyroglutamic acid and the de-polylysine modification of C end for charge isomer, holds pyroglutamic acid and/or C end to take off polylysine modification ratio reduce gradually by peak 1 to peak 4, N; Also part variation is there is, as the Man5 content of peak 4 than peak 1 obviously increases in sugar-type content.Therefore, in conjunction with cation-exchange chromatography, our bright method can be used for detecting the glycosylation of analysis antibody charge isomer and end modified difference.
embodiment 4uPLC-MS method of the present invention is adopted to measure the glycosylation of antibody C (IgG2) and end modified situation
(5 μ g antibody A join in 10 μ L6M guanidine hydrochloride solutions to adopt the reducing condition after optimizing in the present invention, add 0.5MDTT solution 2 μ L again, finally adding appropriate 6M guanidine hydrochloride solution makes DTT final concentration be 50mM, 65 DEG C of reaction 45min), be separated (consistent with embodiment 1.1) through UPLC, ESI-MS detects (consistent with embodiment 1.1) and normalization data process (consistent with embodiment 1.2) and analyzes the glycosylation of antibody C and end modified.Fig. 7 is the chromatogram that antibody C adopts UPLC-MS method of the present invention to record after reduction, and the retention time of light chain (LC) is 6.6min, and 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), and not easily cyclisation forms pyroglutamic acid, light chain or the heavy chain of pyroglutamic acid therefore do not detected; Heavy chain major part there occurs the de-lysine of C end.The sugar-type of antibody C mainly comprises G0F, G1F, Man5, G0 and G2F, and the corresponding molecular weight containing de-lysine heavy chain is 50206Da, 50367Da, 49978Da, 50059Da and 50531Da, consistent with theoretical value; Its content is respectively 58.0%, and 19.5%, 13.7%, 6.6%, 2.2%.Therefore, the inventive method is equally applicable to Immunoglobulin IgG2 glycosylation and end modified detection.
embodiment 5the present invention is adopted to measure the glycosylation of antibody A and the kit method of end modified situation
Kit is made up of reagent A and reagent B, and wherein reagent A is 6M guanidine hydrochloride solution; Reagent B is 0.5MDTT solution.
Use the method for the glycosylation of kit detection antibody A and end modified situation specific as follows:
(protein concentration should be greater than 1 μ g/ μ L to get 20 μ g antibody A, if be 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, at 65 DEG C, react 45min.Reactor product adopts UPLC to be separated (consistent with embodiment 1.1), and ESI-MS detects (consistent with embodiment 1.1) and normalization data process (consistent with embodiment 1.2) and analyzes the glycosylation of antibody A and end modified.Within continuous 5 days, carry out repeating to test (reconfiguring sample and mensuration).
Result shows, and light chain and the heavy chain of antibody A effectively split, and realize baseline separation in chromatogram.As shown in table 5, METHOD FOR CONTINUOUS DETERMINATION 5 days, light chain N holds pyroglutamic acid, and heavy chain N holds the relative standard deviation RSD% of pyroglutamic acid and the de-lysine measured value of C end to be less than 2%; The relative standard deviation RSD% of sugar chain G0F, G1F and G0 relative content measured value is less than 5%, Man5 and G0F-GN is less than 10%.To sum up, this method can realize normalizing operation, and reappearance is good, can be used for setting up measuring immunoglobulin (Ig) glycosylation and end modified kit method.
The reappearance that this method measures the end modified situation of antibody A applied by table 5
Specific description of embodiments of the present invention does not above limit the present invention, and those skilled in the art can make various change or distortion according to the present invention, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (8)
1. measure the glycosylation of immunoglobulin charge isomer and a method for end modified situation, said method comprising the steps of:
1) immunoglobulin (Ig) that protaminase enzyme cuts front and back is analyzed in use cation-exchange chromatography (CEX-HPLC), and collects different immunoglobulin charge isomer by after retention time post;
2) step 1 is made) middle immunoglobulin (Ig) is after denaturant sex change, and use reductive agent reduces, thus splits light chain and heavy chain;
Wherein, described step 2) comprising: the 1-6M aqueous guanidinium-H CL adding 10-30 μ L in a certain amount of immunoglobulin (Ig), 1-4 μ L dithiothreitol (DTT) (DTT) aqueous solution is added after mixing, make immunoglobulin (Ig) generation sex change, reduction reaction, wherein, in reaction solution, DTT final concentration is 25-100mM, and immunoglobulin (Ig) final concentration is 0.2-3 μ g/ μ L;
3) use anti-phase ultrahigh pressure liquid phase chromatrographic separation step 2) in the light chain of immunoglobulin (Ig) and heavy chain;
4) use mass spectroscopy step 3) in the molecular weight of the light chain that obtains and heavy chain; And
5) analytical procedure 3) in chromatographic data and step 4) in mass spectrometric data, thus measure the glycosylation of described immunoglobulin (Ig) and end modified situation;
Described step 2) in immunoglobulin (Ig) generation sex change, reduction reaction temperature be 50-65 DEG C, the reaction time is 45min-120min;
Wherein, step 3) separation condition of described anti-phase ultrahigh pressure liquid phase chromatogram is selected from:
A) chromatographic column: Waters, ACQUITYUPLCcolumn, BEHC4, particle diameter 1.7 μm, aperture
2.1 × 50mm; Chromatographic condition: chromatogram column temperature is set as 55 ~ 65 DEG C, sample size 0.2 ~ 3 μ g; Mobile phase X is 0.1% formic acid water, and mobile phase Y is 0.1% formic acid acetonitrile, and flow velocity is 0.4ml/min; Condition of gradient elution is: 0 ~ 5min, 10%Y; 5 ~ 5.1min, 10 ~ 25%Y; 5.1 ~ 15min, 25% ~ 35%Y; 15 ~ 15.1min, 35% ~ 90%Y; 15.1 ~ 18.0min, 90%Y; 18.0 ~ 18.1min, 90% ~ 10%Y; 18.1 ~ 21.0min, 10%Y;
Or b) chromatographic column: Waters, ACQUITYUPLCcolumn, BEHC4, particle diameter 1.7 μm, aperture
2.1 × 50mm; Chromatographic condition: chromatogram column temperature is set as 60 DEG C, sample size 1 μ g; Mobile phase X is 0.1% formic acid water, and mobile phase Y is 0.1% formic acid acetonitrile, and flow velocity is 0.4ml/min; Condition of gradient elution is: 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.
2. method according to claim 1, 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. method according to claim 1, is characterized in that, described step 2) in DTT final concentration be 50mM.
4. method according to claim 1, is characterized in that, described step 2) in immunoglobulin (Ig) generation sex change, reduction reaction temperature be 65 DEG C, the reaction time is 45min.
5. method according to claim 1, is characterized in that, described step 3) comprising: adopt C4 reverse ultrahigh pressure liquid phase chromatrographic separation step 2) in the light chain of immunoglobulin (Ig) and heavy chain, to realize the baseline separation of described light chain and heavy chain.
6. method according to claim 1, is characterized in that, described step 4) comprising:
Adopt electrospray ionization mass spectrometry determination step 3) in the molecular weight of the light chain that obtains and heavy chain, wherein at 0-5min, stream leads to waste liquid, 5-16min, and stream leads to mass spectrum, then adopts positive ion mode to gather mass spectrometric data;
Mass Spectrometry Conditions is set as:
Taper hole air-flow is 50.0L/Hr, and desolvation gas is 800.0L/Hr, and desolventizing temperature is 500 DEG C, and taper hole voltage is 20-40V, and sweep limit is 400-2500Da, and sweep time is 1s.
7. method according to claim 1, is characterized in that, described step 5) comprising:
By step 3) in the chromatographic peak area that the obtains N that calculates the light chain of described immunoglobulin (Ig) hold pyroglutamic acid ratio, by step 4) in the mass spectrometric data that obtains calculate the sugar-type relative content of the heavy chain of described immunoglobulin (Ig) and N holds pyroglutamic acid and the de-lysine ratio of C end.
8. method according to claim 1, is characterized in that, described immunoglobulin (Ig) is human immunoglobulin(HIg).
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| Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics;Douglas S. Rehder等;《Journal of Chromatography A》;20051117;第1102卷;第164页首段，第165页2栏首段，3段，第166页首句，2.1节，2.2.1节，2.3.1节，第167页2.4.1节，第168页2栏2段，图5，第169页首段1-4行，2栏首段倒数1-5行，第170页3.4节标题，首段，第171页表3 * |
| 新型抗CD2人源化抗体的表征结构分析;郭清城;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20121015(第10期);第19页标题二，第20页"4.色谱柱"节，第21-24页，第26页图2.2.3，第49页首段 * |
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