| The composition analysis and structure characterization of drugs play an important role in the quality control of drugs.It is concerned with every stage from drug design,drug development,drug production and clinical use.With the continuous technique developing and the encouraging of quality by design concept,higher requirements are put forward for drug quality research.The diversity of drug components and the complexity of macromolecular structure modification are the research difficulties and hot spots in the field of drug analysis.In this study,a comprehensive quality control stretagy based on mass spectrometry technology is developed for Traditional Chinese Medicine?TCM?,and a mass spectrometry study for structural characterization of antibody macromolecular drugs is also developed.In quality control study for TCM,DART-MS method was developed for saponins and oligosaccharides detection in this study,and the methods overcomes the difficulty of ionization to multihydroxyl compounds,as a result saponins and oligosaccharides could be detected and quatified by using DART-MS.By investigate the MS spectrum of methylated saponins and oligosaccharides,the gas phase reactions in DART ion source was summarized,also tandum mass spectrometry were compared with deuteration methylated products and by which fragmentation pathway of methylated saponins and oligosaccharides was proposed.Considering there is multiple active ingredients ginseng including gensenosides,oligosaccharides and amino acids,simultaneous and comprehensive analysis is important and meaningful for ginseng quality control.This study developed a simultaneous quantitative analysis method for the three types of ingredients in ginseng.All three types of compounds could be separated in normal phase liquid chromatography,and extraction condition was also investigated in order to obtain good recovery for three types of compounds.The method was used to compare active ingredients in Asian ginseng and American ginseng,and differentiation markers were found out by using PCA statistical analysis.In the study of disulfide bond characterization,sample used was a bispecific antibody,we were aiming at the disulfide bond mismatch problem which is easy to occur in the bispecific antibody manufacture.Mass spectrometry is powerful for this investigation,a Top-dowm MS including intact MS,sub-unit MS,peptide MS were used for the disulfide linkage determination for a bispesific antibody,a Top-down MS based systematic characterization shows that the investigated bispecific antibody doesn't contain mismatched dislfide impurities.In charge heterogeneity study for the bispecific antibody,strong cation exchange chromatography?SCX?showed that the charge variants of the antibody comprise acidic and basic peaks in addition to the highest proportion of the main peaks.By collecting each charge variant in SCX and structure investigation using liquid chromatography-mass spectrometry,we confirmed that the modification of proteins in the main peak were cyclization of N-terminal glutamine and dissociation of C-terminal lysine.The basic peak corresponded mainly to an undissociated lysine at C-terminal and Glysine cleavage at C-term;fragments,deamidation,glycation as well as thioether in F?ab?'2 were responsible for the acidic peaks.This study showed that LC-MS is suitable for the study of hydrophobic and charge heterogeneity of antibodies.Subunit level analysis combined with structure confirmation at peptide map level is an efficient strategy for structure confirmation.In the study of fragment of antibody,we selected the cases of Fab and Fc domain cleavage.In the case of Fc fragmentation,a case study was performed to identify the cleavages found in a mutated monoclonal IgG1 antibody which was used as a mother mAb for bispecific antibody assembly.The monoclonal antibody employs mutated N297 in Fc.However,it exhibited weak stability as easy to form fragments and aggregates,indicating the mutations strongly impact stability.Further studies demonstrated cleavages were identified to occur in Fc domain with two hot spots,and the cleavages were convinced to be induced by Fc deglycosylation by comparing with deglycosylated mAb.In the case of Fab cleavage study,we analyzed the Fab region cleavage of IgG1antibody caused by high temperature.Through high temperature destruction experiment,combined with CE-SDS and mass spectrometry analysis,we identified that the cleavage of the antibody occurred at the 55th-56th amino acid site of the heavy chain N-terminal in Fab region.Also,we proposed the fragmentation mechanism,suggested that the cleavage was caused by the serine in the X-Ser hotspot sequence,hydroxyl group attacks the adjacent NH2,followed by rearrangement and hydrolysis.This study shows that LC-MS can effectively solve the problem of structural confirmation of antibody fragments and effectively screen and identify the fragmentation sites.In summary,this study established the quality control strategy of multi-component simultaneous analysis of TCM by using mass spectrometry.DART-MS showed the advantage of multi-component rapid analysis in profiling the composition of TCM,which can be used for the rapid evaluation,while NPLC-MS realized the qualitative and quantitative analysis detection of saponins,oligosaccharides and amino acids comprehensively.The two methods effectively improved the quality evaluation efficiency and accuracy of TCM.In facing the characterization challenge brought by the diversity of macromolecular structural modification,the mass spectrometry technology based Top-down analysis strategy,namely sreucture analyze on the levels of intact molecule,subunit and peptide map,showed the advantages of high analysis efficiency,accurate hot spot identification and comprehensive modification screening,and were successfully used for charge heterogeneity analysis,trunction site screening and disulfide link confirmation of macromolecules drug.In a word,mass spectrometry can be seemed as a powerful tool for the quality control of TCM and the structural characterization of macromolecular drugs. |
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