Quantitation Methods Development And Application For N-glycosylation Of Immunoglobulin By Mass Spectrometry

Glycans has been recognized as an emerging biomolecules carried important biological information to follow DNA and proteins. It is an extension of “DNA-mRNA-Protein” genetic line. In the multicellular organism, glycosylation as an important post-translational modification played a key part in vivo. Because glycans are not the direct production of genes and their structures are diversity, it is hard to systematically analyze and study the biological function of them. To know more about glycans, the study of glycomics was came up.Glycomics has become a new frontier in the post of genomics and proteomics. Researchers use it to illustrate the function of genes and cells by studying the glycans and glycopeptides/glycoproteins in vivo. Glycosylation is a normal phenomenon existing between the tumor formation and development. It is very important to do a deeper research on glycosylation modification for the study of the tumor mechanisms, diagnose and therapy. By analyzing the glycoproteins differences between the tumor and normal cells could provide a meaningful information for the early diagnosis of tumor.This study is completed in the Lebrilla B. Carlito lab in the University of California, Davis. They have developed an UPLC-QqQ-MS/MS method to examine quantitative changes in glycosylation at a site-specific level on immunoglobulin G, immunoglobulin A and immunoglobulin M. Compared with the traditional glycans quantification methods, the new method has the following advantages. Firstly, the amount of sample was reduced. The glycopeptides only accounts for 2~5% of the total protein, the amount of the released glycans was even less. Moreover, the signal of glycans in the mass was very low and hard to pick up. So a larger amount of samples was needed to get the good abundance. This new approach was to quantify the glycopeptides/glycoproteins, which means the glycan and its conjunct peptide were assayed together, as a result the amount of the substance to be determined was increased and the signal was better for the glycopeptides in mass. The sampling amount was decreased from 50μL to 2μL in this new approach. Secondly, the new approach was costsaving, convenient and stable. There is no need to release N-glycans in the new method, so the related procedures such as SPE and evaporating were abolished. Moreover, without the release of N-glycans, the unrepeatable quantification results which is due to the incompletable enzymatic reaction were overcome. Lastly, the new approach provides information regarding both the absolute amount of protein and the site-specific glycosylation profiles and will thus be useful to determine if altered glycosylation profiles in serum are due to a change in protein glycosylation or a change in protein concentration. The results showed that this method was easy, reliable and repeatable, it will have considerable value in the disease biomarker discovery.Based on the glycopeptides quantification methods built by this group, we did a research on the glycopeptides biomarker analysis of gastric cancer. The serum samples of gastric cancer and gastritis were separated into two groups randomly, and the two groups were analyzed together. The glycopeptides of IgG, IgA and IgM were analyzed and quantified by the above method. The data of first group were analyzed using t-test. According to the data analysis of the first group, the glycopeptides with significant differences between gastric cancer and gastritis were found as the biomarker, and then they were applied to the second group by using the Partial Least Squares method from R Language statistics software to check whether the gastric cancer and the gastritis group could be separated by these glycopeptides. The results showed that seven glycopeptides of significant differences were found among about sixty different glycopeptides, and the gastric cancer group could almost be separated from gastritis group.In this thesis, we not only did the research on the cancer disease but also the related therapy drugs. Recombinant monoclonal antibody( rMAb) drugs as an effective biopharmaceutical for cancer and other chronic diseases have been paid high attention from their emergence. This is due to the specificity of these drugs toward target antigens with the functions of activating the immune system to kill tumor cells, blocking the signal transduction of tumor cells to proliferate, and carrying cancer drugs of radiation targets to tumor cells. Currently, there are more than 30 approved rMAb drugs and hundreds of new rMAb drug candidates under clinical trials. So the outstanding performance of this kind of drugs make it valuable in development. Today, the focus on discovery and development of rMAb drugs continues to grow rapidly within the pharmaceutical industry. To date, all licensed rMAbs have been of the immunoglobulin G(IgG) class; however, four subclasses of IgG(IgG1, IgG2, IgG3 & IgG4) also exhibit unique effector functions. Therefore, it is important to select the IgG subclass that is anticipated to have the most potent activity for a given disease. Further studies have shown that glycosylation of IgG influences both its physiochemical properties and, more importantly, its cell-mediated effector functions such as complement binding and activation. Clearly, in manufacturing of therapeutic recombinant monoclonal antibody, the site specific compositions of N-glycosilation and assessment of Nglycan site occupancy are of utmost importance. This growth is accompanied with new challenges in quality control and analytical characterization during drug development and production. Consequently, there is an urgent demand for developing high-performance analytical techniques for characterization of N-glycosylation and quantify the N-glycan site occupancy of rMAbs.In this study, we utilized the power of MRM to observe and quantify N-glycopeptides directly from rMAbs without protein enrichment and N-glycan release. The N-glycopeptides frofile of rMAbs was built and glycopeptides of the rMAbs could be quantified fast and reliable. The results showed the method we built was convenient, fast and repeatable. Based on the approach we developed, the glycopeptides of six approved rMAbs were analyzed, the results showed that no matter the subclasses of rMAbs, the main glycans and the percentage of the glycans were almost the same.For the further study, we developed the N-glcan occupancy quantification method for rMAbs. When N-glycans were released from glycopeptides by PNGase F, the peptides residue will change from Asparagine to Aspartic Acid with the increase of the peptide molecular weight by 0.984 Da. The N-glycans site occupancy could be quantified according to this change by using MRM method. After the regression calculation by the peptides standards, the N-glycans occupied and unoccupied peptides could be quantified. The results of N-glycans occupancy analysis of the six rMAbs showed that all these six drugs were highly glycosylated with the N-glycan site occupancy over 97%.In this study, we built the fast, efficient, accurate and robust MRM methods for the quantitation of N-glycopeptides in the study of gastric cancer and rMAb drugs. It is of great meaning for the further study of searching the biomarkers of diseases, and the development of rMAbs.