| Glycan modification of glycoproteins includes glycosylation and glycation,which have important effects on the safety,stability,immunogenicity and biological activity of glycoproteins,and are closely related to the occurrence and development of many diseases.Therefore,the characterization of glycan modification on glycoprotein drug has important biological significance.Due to the complexity of the structure of glycan modification in different glycoproteins,currently the comprehensive research on glycan modification is still a huge challenge.Therefore,it is necessary to develop a comprehensive strategy to characterize the glycan modification for different glycoproteins.In this paper,the glycosylation and glycation of complex glycoproteins and their effects on charge heterogeneity were investigated from the level of intact glycoprotein,the glycan chains separated from protein and glycopeptide.In the first chapter,we introduced the status of glycoprotein drugs,the classification of glycan modification and its importance to glycoprotein drugs first of all.Secondly,we reviewed the commonly used research methods for determine the content of glycosylation,glycosylation site,glycan type and strategies for glycation modification analysis.The second chapter,using BSA as the standard to establish the"quasi-internal standard method",The RSD of multi-charge peak of BSA was less than or equal to 0.075%and the RSD between batches was between 0.001%and 0.004%,the method error<0.01%.The results showed that the method has good accuracy and high reliability.The relative content of N-glycan,sialic acid of O-glycan,O-glycan and glycation was accurate determination by this method.The biosimilars were 24.6%,2.9%,9.0%and5.1%,respectively.The original drugs were 25.6%,2.9%,7.9%and 3.5%respectively.This method not only ruled out the interference of standards but also improved the determination accuracy,which has obvious advantages in the consistency analysis applied to complex glycoproteins.The main N-glycan of all 8 McAbs were G0F/G0F and G0F/G1F based on the molecular weight of intact glycoprotein.The hexosaccharide index?HexI?was between 0.2 and 1.6.The lower HexI,the higher the relative proportion of G0F/G0F.The higher the Hex I,the lower the relative proportion of G0F/G0F and the relative content of G0F/G1F,G1F/G1F and G1F/G2F increased.The hexose index could well reflect the basic glycoform distribution of the antibody drugs,and can be used as an important parameter for the optimization of fermentation process and the quality control of glycoforms.In addition,we proposed the concept of sialic acid index?SAI?and applied to analysis the sialic acid and charge variant.The SAIs of three batches of monoclonal antibodies ranged from 0.016 to 0.018 and the consistency between batches was good.The new concept and method for analyzing the consistency of intact glycoproteins described above are of important reference value for the rapid quality evaluation of glycoproteins.The third chapter,the analysis of the glycosylation site of complex glycoprotein.In this study,N-glycosylation and O-glycosylation sites of recombinant human erythropoietin?rhEPO?and rhTPO were analyzed from glycopeptide levels using tandem mass spectrometry.Three N-glycosylation sites?N24,N38,N83?and one O-glycosylation site?S126?of rhEPO were successful identificated in a score of more than700.The identification results were reliable.The result of glycosylation rate at each locality showed that N38 was the highest,N24 was the next,N83 was the lowest analyzed baased on both the"detection times"and"ionic strength",and the total glycosylation rates were both higher than 65%.The agreement between methods and batches were good.Five N-glycan sites?N176,N185,N213,N234 and N327?of rhTPO were successfully identified.The identification scores were all more than 300,which were all very reliable.The highest rates of N176 and N185 were confirmed by the two methods.Followed by N234 and N327,while N213 was the lowest,total glycosylation rate was more than 75%,good agreement between methods and batches.In addition,57 O-glycosylation sites were obtained according to rhTPO O-glycoforms,and the relative ratio of S106 was 49%,which was significantly higher than other sites and was the most important O-glycosylation site of rhTPO.Glycosylation rate analysis provides a simple and effective method for the batch-to-batch alignment of complex glycoproteins.In addition,this study also successfully identified the N-glycosylation site N298 by O18 labeling,scoring 949,the identification results are very reliable,and the method effectively avoided the false positive due to the deamination caused by the alkaline environment and was favorable for the subsequent research on the glycosylation sites of complex glycoproteins.In the fourth chapter,we introduced the glycan analysis strategies of monoclonal antibody drugs and complex glycoproteins in the level of glycan chains and glycopeptide.Firstly,22 N-glycan glycoforms were successfully assigned to the glycan chain according to the GU value,and the relative content of the common glycoform was more than 98%.The consistency among batches was good.Among them,the relative content of G0F was highest,followed by G1F.Eleven peaks were identified by UPLC-MS/MS,corresponding to 11 antibody glycoforms.Then,using the optimized glycan screening method,139 N-glycan glycoforms of 5 monoclonal antibodies were detected at the glycopeptide level,among which there were 30 common glycoforms.The relative content of G0F was the highest among the 5 kinds of antibodies.As the main glycoform,it was consistent with the results of glycan chain analysis.Finally,we analyzed the glycoprotein of rhTPO at the level of glycopeptide,and identified 226 N-glycans and 75 O-glycans,of which there were 22 mainly N-glycans and 26 mainly O-glycans,batch consistency was good.This study provided a more comprehensive research strategy for the analysis of glycoforms of monoclonal antibodies and complex glycoproteins.The fifth chapter,Glycation is a protein glycan modification caused by chemical reaction,and closely related to the process,should generally be avoided,there were small number of foreign reports and no reports of domestic.A glycation analysis strategy was developed to detect the glycosylated antibody from the sample collected to ultra-high resolution mass spectrometry.The hexose index of glycated sample was about 1 larger than the conventional sample analysis from two aspects of the intact molecule and the enzymatic peptide,and the glycated sample was modified with 1 to 2 lysine?K?,100/150/170 K was the main glycosylation site in light chain,while the heavy chain also existed arginine?R?and N-terminal glycation,325 K and N-terminal of heavy chain prone to glycated.This study can not only play an important role in the characterization and evaluation of the quality of monoclonal antibodies,but also help the development of monoclonal antibody drug culture technology,formulation and preparation technology,and provide new ideas for the study of drug efficacy and function of monoclonal antibodies.The sixth chapter,the charge variants?acid component,main component and basic component?of IgG1 and IgG2 were collected by cation exchange chromatography?CEX?.Ultrahigh resolution mass spectrometry was used to analyze the charge heterogeneity at two levels of intact molecules and digested peptides.For IgG1,12 glycoforms were identified from intact glycoprotein molecules,of which 7 were detected in 3 components and the relative content were the same,which could eliminate the influence of basic glycoforms.All five glycoforms had sialic acid.According to the SAI analysis,the more the number of sialic acid at the glycan chain terminal,the higher the sialic acid index and the more acidic the protein,the sialic acid was one of the major sources of charge heterogeneity.Eight MS peaks were detected from the N-glycan removed antibody and pyroglutamate?PyroQ?occurred at the N-terminal Q.Therefore,N-terminal PyroQ was not responsible for charge heterogeneity.The glycation content of the two acidic components was slightly larger than that of the main component,but the difference was insignificant.Glycation may be one of the sources of charge heterogeneity.Deamidation and glycation were slightly higher in acidic components and may be one of the reasons for acidic components but not one of the primary causes for charge heterogeneity analysis at the peptide level.There was almost no contribution from oxidation and N-terminal pyroglutamylation,and the modification rate of the major modification sites of the light and heavy chains has a slight difference between the components,which may have a weak influence on the charge heterogeneity.For IgG2,isoelectric point capillary electrophoresis before and after glycan removed showed that N-glycan modification was not the main reason leading to its charge heterogeneity.The results showed that the relative ratio of IgG2-A disulfide bonds in Peak3was the highest,and the relative proportion of IgG2-B disulfide bonds showed a decreasing trend between Peak1 and Peak3.The relative ratio of IgG2-A/B disulfide bonds?A/B?was slightly different between the two statistical methods,indicating that disulfide bonds were the major cause of denosumab charge variant.This study provided a complete method for the analysis of charge variants and an important scientific basis for screening and quality control of new drugs. |
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