CN105628927A - Fusion protein molecular weight analysis method - Google Patents
Fusion protein molecular weight analysis method Download PDFInfo
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Abstract
The present invention discloses a fusion protein molecular weight analysis method, and specifically relates to a method for carrying out complex glycosylated fusion protein accurate molecular weight analysis by using ESI-Q-TOF, particularly to a sample pre-treatment method for removing fusion protein N-glycan and sialic acid by using specific glycan removing enzyme, wherein the treated fusion protein is separated through a salt removing column, the effluent is broken through an ESI source to obtain continuous multi-charged ions, and tandem Q-TOF mass spectrometry analysis and deconvolution calculation are performed to obtain the complete protein accurate molecular weight of the O-glycan core isomer. With the method of the present invention, the high inhibition of the N-glycan and/or sialic acid on the fusion protein ionization efficiency can be overcome, and the complete protein molecular weight of the O-glycan fusion protein can be accurately determined.
Description
Technical field
The invention belongs to biomedicine technical field, more particularly to a kind of protein molecular component analysis method.
Background technology
Cytokine recombination fusion protein is that a class utilizes engineered method the Codocyte factor and other are had the protein molecular gene order of specific function to connect and express respective egg white matter fusion product. its construction features is merged in the active territory in cytokine function activity territory with other molecules, and each component can play synergism, makes the more each monomer of biologic activity of fusion protein be greatly enhanced. fusion protein is glycosylation modified to be all closely related with its biological activity, structure and pharmacokinetics, also can directly or indirectly affect it to the resistance of protease and the combination of Fc receptor and the interaction of C1Q. component, Half-life in vivo, related biological functional effect etc. specifically glycosylation modified participate in important IC effector function, also can by affecting serum half-life (RajuTS with the combination of newborn Fc gamma receptor, ScallonBJ.GlycosylationintheFcdomainofIgGincreasesresist ancetoproteolyticcleavagebypapain.BiochemicalBiophysical ResearchCommunications2006, 341:797-803) (WrightA, SatoY, OkadaT, ChangK, EndoT, MorrisonS.InvivotraffickingandcatabolismofIgG1antibodies withFcassociatedcarbohydratesofdifferingstructure.Glycob iology2000, 10:1347-1355).
Glycosylation modified is highly important to fusion protein biological function, but the glycosylation modified of complexity brings the challenge bigger relative to general IgG1 antibody often to protein structure phenetic analysis. Monomer Fc as each in Embrel part has 1 N-sugar site, Fab part has 2 N-sugar sites, additionally according to the literature possibly together with 13 O-sugar site (HouelS, HilliardM, YuY, McLoughlinN, MartinSM, RuddPM, etal.N-andO-GlycosylationAnalysisofEtanerceptUsingLiquid ChromatographyandQuadrupoleTime-of-FlightMassSpectrometr yEquippedwithElectron-TransferDissociationFunctionality. AnalyticalChemistry2013; 86:576-584). The each monomer Fc part of rhCTLA4-Ig also has 1 N-sugar site, Fab part has 2 N-sugar site (BongersJ, DevincentisJ, FuJ, HuangP, KirkleyDH, LeisterK, etal.Characterizationofglycosylationsitesforarecombinant IgG1monoclonalantibodyandaCTLA4-Igfusionproteinbyliquidc hromatographymassspectrometrypeptidemapping.JournalofChr omatographyA2011; 1218:8140-9), additionally there are 2 O-sugar sites. Albumen can be characterized by molecular weight analyse on a macro level, can confirm that whether protein expression is correct at molecular weight levels, simultaneously it can be identified that the different isomerization body of albumen modifies form and ratio, provide clue and foundation intuitively for further confirming that albumen changes in peptide fragment level. Owing to fusion protein intact proteins Ionization Efficiency is suppressed fairly obvious by the N-sugar on sialic acid and Fab section, general bibliographical information all adopts enzyme-specific such as Ides and papain Papain that after scaling off separation complete monomer, Fc section part is carried out molecular weight analyse (TanQ again, GuoQ, FangC, WangC, LiB, WangH, etal.Characterizationandcomparisonofcommerciallyavailabl eTNFreceptor2-Fcfusionproteinproducts.MAbs2012; 4:761-74); (LynaughH, LiHandGongB.RapidFcglycosylationanalysisofFcfusionswithI deSandliquidchromatographymassspectrometry.MAbs2013; 5:761-74).
Therefore, how effectively fusion protein is carried out intact protein molecules component analysis and be still solving the technical problem that of current urgent need.
Summary of the invention
The present invention provides a kind of fusion protein entire molecule component analysis method, the suppression of Ionization Efficiency when the method overcoming the sugar of N-in prior art and sialic acid to fusion protein intact protein molecules component analysis, can remove after N-sugar and sialic acid the molecular weight of the fusion protein containing O-sugar core by Accurate Determining. The present invention is achieved by the following technical solutions, comprises the steps: 1, fusion protein N-sugar (N-Glycosylations) removes; 2, fusion protein sialic acid (SialicAcid) is removed; 3, ESI-Q-TOFMS analyzes, Deconvolution calculation. Wherein, step 1 and 2 sequencings can exchange.
More specifically, the invention discloses:
A kind of fusion protein molecule analysis method, described method includes step: (1) fusion protein N-sugar is removed and/or sialic acid is removed; (2) to removing, N-is sugared and/or sialic fusion protein carries out molecular weight analyse.
Further, a kind of fusion protein molecule analysis method of disclosure of the invention, it is characterized in that, said method comprising the steps of: (1) fusion protein N-sugar is removed, (2) fusion protein sialic acid is removed, and (3) carry out molecular weight analyse to removing the sugared and sialic fusion protein of N-; Wherein, above-mentioned steps (1) and (2) order can be exchanged.
Preferably, when fusion protein N-sugar is removed, solution ph is 7.0��8.0; When sialic acid is removed, solution ph is 4.0��7.0. It is highly preferred that described pH value is to be regulated by acid or alkaline volatile solution.
In certain embodiments, described fusion protein N-sugar is removed and is realized by the following method: take fusion protein sample, adds buffer (such as: NH4HCO3Or 1%NH3.H2O) regulate pH value to 7.0��8.0, add Peptide N-glycosidase F, after mixing, hatch 24 hours for 37 DEG C.
In certain embodiments, described fusion protein sialic acid is removed and is realized by the following method: take fusion protein sample, adds buffer (such as: NH4HCO3Or 1%FA (FA: formic acid)) regulate pH value to 4.0��7.0, add sialidase, hatch 24 hours for 37 DEG C.
Preferably, the molecular weight of described fusion protein is to be analyzed by ESI-Q-TOFMS method.
In certain embodiments, described ESI-Q-TOFMS analyzes and adopts following methods to realize: by fusion protein 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then desalting column (preferred MassPREPTMMicroDesaltingColumn, 20 ��m, 2.1 �� 5mm, Waters company) desalination; Finally carry out mass spectral analysis.
In some embodiments 1��7, described fusion protein is Etanercept, and fusion protein described in embodiment 8 is rhCTLA4-Ig.
Test result indicate that, by utilizing specificity desaccharide enzyme again fusion protein to be analyzed after removing fusion protein N-sugar and/or sialic acid, can obtain containing O-sugar core fusion protein accurate molecular masses. additionally, experimental result also shows, only carry out N-sugar to be removed without carrying out sialic acid removal or only carry out sialic acid being removed without carrying out N-sugar removal such as fusion protein to be analyzed, fusion protein Ionization Efficiency when the fragmentation of ESI source is subjected to higher suppression, finally can only obtain a few containing O-sugar core fusion protein accurate molecular masses, as utilized specificity desaccharide enzyme both to remove N-sugar, also removing sialic acid, fusion protein Ionization Efficiency when the fragmentation of ESI source suppresses to be substantially reduced, and may finally obtain most containing O-sugar core fusion protein accurate molecular masses. additionally, test result indicate that, fusion protein N-sugar carries out after removing not regulating pH of cushioning fluid when sialic acid is removed again, and carry out again after sialic acid removal not regulating pH of cushioning fluid when N-sugar is removed, fusion protein Ionization Efficiency when the fragmentation of ESI source has been also affected by certain suppression, can obtain mainly several containing O-sugar core fusion protein accurate molecular masses, but effect is not as regulating the mode of pH of cushioning fluid, carry out again after removing such as fusion protein N-sugar regulating pH of cushioning fluid 4.0��7.0 when sialic acid is removed, and carry out again after sialic acid removal regulating pH of cushioning fluid 7.0��8.0 when N-sugar is removed, fusion protein Ionization Efficiency when the fragmentation of ESI source suppresses to be substantially reduced, can obtain most containing O-sugar core fusion protein accurate molecular masses.
In a word, compared with prior art, the present invention has following beneficial effect: the present invention can directly Accurate Determining fusion protein containing O-sugar core isomer molecule amount.
Accompanying drawing explanation
Fig. 1, schemes for the embodiment of the present invention 1 fusion protein typical case multi-charge TIC;
Fig. 2, for the embodiment of the present invention 1 fusion protein deconvoluting intact protein molecules component analysis result figure;
Fig. 3, schemes for the embodiment of the present invention 2 fusion protein typical case multi-charge TIC;
Fig. 4, for the embodiment of the present invention 2 fusion protein deconvoluting intact protein molecules component analysis result figure;
Fig. 5, for sialic acid deconvoluting intact protein molecules amount relative analysis result figure de-under pH4.0 and pH5.0 condition in the embodiment of the present invention 3;
Fig. 6, for sialic acid deconvoluting intact protein molecules amount relative analysis result figure de-under pH5.0 and pH6.0 condition in the embodiment of the present invention 3;
Fig. 7, for sialic acid deconvoluting intact protein molecules amount relative analysis result figure de-under pH6.0 and pH7.0 condition in the embodiment of the present invention 3;
Fig. 8, for the embodiment of the present invention 4 fusion protein deconvoluting intact protein molecules component analysis result figure;
Fig. 9, for the embodiment of the present invention 5 fusion protein deconvoluting intact protein molecules component analysis result figure;
Figure 10, schemes for the embodiment of the present invention 6 fusion protein typical case multi-charge TIC;
Figure 11, for the embodiment of the present invention 6 fusion protein deconvoluting intact protein molecules component analysis result figure;
Figure 12, schemes for the embodiment of the present invention 7 fusion protein typical case multi-charge TIC;
Figure 13, for the embodiment of the present invention 7 fusion protein deconvoluting intact protein molecules component analysis result figure;
Figure 14, for the embodiment of the present invention 8 fusion protein (rhCTLA4-Ig) typical case multi-charge TIC figure.
Figure 15, for the embodiment of the present invention 8 fusion protein (rhCTLA4-Ig) deconvoluting intact protein molecules component analysis result figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further, should not be understood as limitation of the present invention. The experimental technique of unreceipted actual conditions in the following example, be all conventionally condition or manufacturer it is proposed that condition test. Fusion protein described in embodiment 1��7 is Etanercept.
Embodiment 1: fusion protein entire molecule component analysis (first N-sugar is removed, and then sialic acid is removed)
Fusion protein intact molecular weight analytical procedure:
(1) N-sugar (N-Glycosylations) removes
Take 50 �� gEtanercept fusion protein sample (pfizer inc, as follows), add 50mMNH4HCO3(pH8.0) solution is to volume 20 �� L, adds the 1 �� L4 times Peptide N-glycosidase PNGaseF (NEB, 500000u/mL) diluted, after mixing, hatches 24 hours for 37 DEG C.
(2) sialic acid (SialicAcid) is removed
The sample will hatched in (1), regulates pH value to 5��6 with 1%FA, adds sialidase Neuraminidase (Roche, 1U/100 �� L) 0.5 �� L, hatch 24 hours for 37 DEG C.
(3) ESI-Q-TOFMS analyzes
The fusion protein of the de-N-sugar of de-sialic acid adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalting column (Waters company, as follows) desalination, mass spectral analysis.
Experimental result: fusion protein typical case multi-charge TIC figure is shown in accompanying drawing 1, and deconvoluting intact protein molecules component analysis result is shown in accompanying drawing 2.
Embodiment 2: fusion protein entire molecule component analysis (first sialic acid is removed, and then N-sugar is removed)
Fusion protein intact molecular weight analytical procedure:
(1) sialic acid is removed
Take 50 �� gEtanercept fusion protein sample, add 50mMNH4HCO3(pH8.0) solution is to volume 20 �� L, regulates pH value to 5��6 with 1%FA, adds sialidase Neuraminidase0.5 �� L, hatch 24 hours for 37 DEG C.
(2) N-sugar is removed
The sample will hatched in (1), uses 1%NH3.H2O regulates pH value to 7��8, adds the 1 �� L4 times PNGaseF (NEB, 500000u/mL) diluted, hatches 24 hours for 37 DEG C.
(3) ESI-Q-TOFMS analyzes
De-N-sugar takes off sialic fusion protein and adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalting column desalination, last mass spectral analysis.
Experimental result: fusion protein typical case multi-charge TIC figure is shown in accompanying drawing 3, and deconvoluting intact protein molecules component analysis result is shown in accompanying drawing 4.
Embodiment 3: de-sialic acid deconvoluting intact protein molecules component analysis contrast under condition of different pH after de-N-sugar
Fusion protein intact molecular weight analytical procedure:
(1) N-sugar is removed
Take 400 �� gEtanercept fusion protein sample, add 50mMNH4HCO3(pH8.0) solution is to volume 160 �� L, adds 2 �� LPNGaseF, after mixing, hatches 24 hours for 37 DEG C.
(2) sialic acid is removed
The sample hatched in (1) is taken 4 parts, every part of 50 �� g, regulate pH value respectively with 1%FA to 4,5,6,7, all add sialidase Neuraminidase0.5 �� L, hatch 24 hours for 37 DEG C.
(3) ESI-Q-TOFMS analyzes
De-N-sugar adopts 50mMNH with de-sialic fusion protein4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalination, mass spectral analysis.
Experimental result: after de-N-sugar, under condition of different pH, de-sialic acid deconvoluting intact protein molecules amount relative analysis result is shown in accompanying drawing 5-7.
Embodiment: 4:N-sugar does not regulate pH of cushioning fluid and carries out the sialic acid removal impact on fusion protein entire molecule component analysis after removing
Fusion protein intact molecular weight analytical procedure:
(1) N-sugar is removed
Take 50ugEtanercept fusion protein sample, add 50mMNH4HCO3(pH8.0) solution is to volume 20 �� L, adds the 1 �� L4 times PNGaseF diluted, and after mixing, hatches 24 hours for 37 DEG C.
(2) sialic acid is removed
The sample will hatched in (1), adds sialidase Neuraminidase0.5 �� L, hatches 24 hours for 37 DEG C.
(3) ESI-Q-TOFMS analyzes
De-N-sugar takes off sialic fusion protein and adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalination, mass spectral analysis.
Experimental result: fusion protein deconvoluting intact protein molecules component analysis result is shown in accompanying drawing 8.
Embodiment 5: sialic acid does not regulate pH of cushioning fluid after removing and carries out the N-sugar removal impact on fusion protein entire molecule component analysis
Fusion protein intact molecular weight analytical procedure:
(1) sialic acid is removed
Take 50 �� gEtanercept fusion protein sample, add 50mMNH4HCO3Solution, to volume 20 �� L, adds sialidase Neuraminidase0.5 �� L, hatches 24 hours for 37 DEG C.
(2) N-sugar is removed
The sample will hatched in (1), adds the 1 �� L4 times PNGaseF diluted, and hatches 24 hours for 37 DEG C.
(3) ESI-Q-TOFMS analyzes
The fusion protein of the de-N-sugar of de-sialic acid adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalination, mass spectral analysis.
Experimental result: fusion protein deconvoluting intact protein molecules component analysis result is shown in accompanying drawing 9.
Embodiment: 6: do not carry out sialic acid and remove the impact on fusion protein molecule component analysis
Fusion protein intact molecular weight analytical procedure:
(1) N-sugar is removed
Take 50 �� gEtanercept fusion protein sample, add 50mMNH4HCO3Solution, to volume 20 �� L, adds the 1 �� L4 times PNGaseF diluted, and after mixing, hatches 24 hours for 37 DEG C.
(2) ESI-Q-TOFMS analyzes
The fusion protein of de-N-sugar adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalination, mass spectral analysis.
Experimental result: fusion protein typical case multi-charge TIC figure is shown in accompanying drawing 10, and deconvoluting intact protein molecules component analysis result is shown in accompanying drawing 11.
Embodiment 7: do not carry out the N-sugar removal impact on the molecular weight analyse of fusion protein
Fusion protein intact molecular weight analytical procedure:
(1) sialic acid is removed
Take 50 �� gEtanercept fusion protein sample, add 50mMNH4HCO3Solution, to volume 20 �� L, adds sialidase Neuraminidase0.5 �� L, hatches 24 hours for 37 DEG C.
(2) ESI-Q-TOFMS analyzes
De-sialic fusion protein adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalination, mass spectral analysis.
Experimental result: fusion protein typical case multi-charge TIC figure is shown in accompanying drawing 12, and deconvoluting intact protein molecules component analysis result is shown in accompanying drawing 13.
Embodiment 8: fusion protein rhCTLA4-Ig entire molecule component analysis (first N-sugar is removed, and then sialic acid is removed)
Fusion protein intact molecular weight analytical procedure:
(1) N-sugar is removed
Take 50 �� grhCTLA4-Ig fusion protein sample (Bristol Myers Squibb company limited), add 50mMNH4HCO3Solution, to volume 20 �� L, adds the 1 �� L4 times PNGaseF diluted, and after mixing, hatches 24 hours for 37 DEG C.
(2) sialic acid is removed
The sample will hatched in (1), adds sialidase Neuraminidase0.5 �� L, hatches 24 hours for 37 DEG C.
(3) ESI-Q-TOFMS analyzes
De-sialic rhCTLA4-Ig fusion protein adopts 50mMNH4HCO3(pH8.0) solution dilution is to 1mg/mL; Then after MassPREPTMMicroDesaltingColumn2.1 �� 5mm desalination, mass spectral analysis.
Experimental result: rhCTLA4-Ig fusion protein typical case multi-charge TIC figure is shown in that the typical intact protein molecular component analysis result of deconvoluting of accompanying drawing 14 is shown in accompanying drawing 15.
To sum up, compared with prior art, the present invention can directly Accurate Determining fusion protein containing O-sugar core isomer molecule amount. additionally, the experimental result of the experimental result of embodiment 6 or 7 and embodiment 1 or 2 contrasts, can be seen that, fusion protein only carries out N-sugar and is removed without carrying out sialic acid removal or only carry out sialic acid being removed without carrying out N-sugar removal, fusion protein Ionization Efficiency when the fragmentation of ESI source is subjected to higher suppression, finally can only obtain a few containing O-sugar core fusion protein accurate molecular masses, as utilized specificity desaccharide enzyme both to remove N-sugar, also removing sialic acid, fusion protein Ionization Efficiency when the fragmentation of ESI source suppresses to be substantially reduced, and may finally obtain most containing O-sugar core fusion protein accurate molecular masses. additionally, embodiment 3, the experimental result of the experimental result of 4 or 5 and embodiment 1 or 2 contrasts, can be seen that, fusion protein N-sugar carries out after removing not regulating pH of cushioning fluid when sialic acid is removed again, and carry out again after sialic acid removal not regulating pH of cushioning fluid when N-sugar is removed, fusion protein Ionization Efficiency when the fragmentation of ESI source has been also affected by certain suppression, can obtain mainly several containing O-sugar core fusion protein accurate molecular masses, but effect is not as regulating the mode of pH of cushioning fluid, carry out again after removing such as fusion protein N-sugar regulating pH of cushioning fluid 4.0��7.0 when sialic acid is removed, and carry out again after sialic acid removal regulating pH of cushioning fluid 7.0��8.0 when N-sugar is removed, fusion protein Ionization Efficiency when the fragmentation of ESI source suppresses to be substantially reduced, can obtain most containing O-sugar core fusion protein accurate molecular masses.
Claims (10)
1. a fusion protein molecule analysis method, it is characterised in that described method includes step:
(1) fusion protein N-sugar is removed and/or sialic acid removal;
(2) to removing, N-is sugared and/or sialic fusion protein carries out molecular weight analyse.
2. method according to claim 1, it is characterised in that said method comprising the steps of:
(1) fusion protein N-sugar is removed,
(2) fusion protein sialic acid is removed,
(3) the removal sugared and sialic fusion protein of N-is carried out molecular weight analyse;
Wherein, above-mentioned steps (1) and (2) order can be exchanged.
3. method according to claim 2, it is characterised in that when fusion protein N-sugar is removed, solution ph is 7.0��8.0.
4. method according to claim 2, it is characterised in that when fusion protein sialic acid is removed, solution ph is 4.0��7.0.
5. method according to claim 3, it is characterised in that described fusion protein N-sugar is removed and is realized by the following method: take fusion protein sample, adds NH4HCO3Or 1%NH3.H2O regulates pH value to 7.0��8.0, adds Peptide N-glycosidase, after mixing, hatches 24 hours for 37 DEG C.
6. method according to claim 4, it is characterised in that described fusion protein sialic acid is removed and is realized by the following method: take fusion protein sample, adds NH4HCO3Or 1% first acid for adjusting pH value to 4.0��7.0, add sialidase, hatch 24 hours for 37 DEG C.
7. method according to claim 1, it is characterised in that the molecular weight of described fusion protein is to be analyzed obtaining by ESI-Q-TOFMS method.
8. method according to claim 7, it is characterised in that described ESI-Q-TOFMS analyzes and includes step: by the NH of fusion protein 50mMpH8.04HCO3Solution dilution is to 1mg/mL; Then desalting column desalination; Finally carry out mass spectral analysis.
9. according to the arbitrary described method of claim 1-8, it is characterised in that described fusion protein is Etanercept or rhCTLA4-Ig.
10. method according to claim 3 or 4, it is characterised in that described pH value is to be regulated by acid or alkaline volatile solution.
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| 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 |
| CN115078728A (en) * | 2021-03-11 | 2022-09-20 | 盛禾(中国)生物制药有限公司 | Rapid analysis method of fusion protein |
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| CN103858003A (en) * | 2011-09-29 | 2014-06-11 | 西雅图基因公司 | Intact mass determination of protein conjugated agent compounds |
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| 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 |
| CN115078728A (en) * | 2021-03-11 | 2022-09-20 | 盛禾(中国)生物制药有限公司 | Rapid analysis method of fusion protein |
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