Applied Research, Bio-mass Spectrometry And Sequence Analysis Of Protein Drugs

After the biological medicine "Biosimilars", their molecular structures need to be characterized in order to make sure the similarity with the original products, and ensure the similar quality, accuracy and efficacy. It is necessary to determine the molecular mass of intact protein, to identify the sequence of amino acids and modifications such as disulfide bond, glycosylation, etc. The research work of this master thesis mainly focuses on the characterization of two kinds of biological medicine with biomass spectrometry, mainly for the determination of intact protein molecular mass and the determination of amino acid sequence as well as its modifications especially with N-glycan chains.The protein used in the second chapter is Recombinant Human Growth Hormone(rhGH) with PEGylation. After the reduction and alkylation response, the sample was analyzed by UPLC-ESI-MS-MS at positive ion mode, which yielded an identification of total 198 peptides from Uniprot database. Then the majority of original raw data from the search results are analyzed and summarized, through such manual evaluation together with the MS spectra, the peptides identified are up to 204, and the sequence coverage is close to 100%. The peptides, abnormally digested by trypsin, are discovered except the normal. Also, the substitution of amino acid and few chemical modifications such as oxidation, deamidated and dehydrated, are observed. Such information plays a very important pole in the characterization of the amino acid sequence of biological drugs. Moreover, it helps to characterize the peptide, the structure of protein, even the related function.The work in the third chapter mainly focuses on the determination of intact N-glycoprotein molecular mass and the analysis of the sequence of N-glycan from the N-glycoprotein. The N-glycoprotein here is Recombinantfollicle stimulating hormone (FSH). The mass values of the two subunits are determined, and the sialic acid is also detected. The sample is unknown N-glycan chain form a drug company. Because of the existence of sialic acid, the UPLC-ESI-MS-MS experiment is done at the negative ion mode. Combined the TIC spectrum, XIC spectra, retention time, MS spectra and MS/MS spectra with the help of software "Analyst", the peaks of glycan fragments are identified. At last the glycanform are obtained successfully through the analysis and calculation on the data.Later, the study was focused to establish a standard method to analyze the sequence of N-glycans, considering that the reducing end derivatization can help to decrease the complexity of mass spectra, especially the fragment ion mass spectra, the work adopt 2-AB(2-amino-benzamideg) as labeling. At the first step, maltohexaose polymerized by six glucoses are used in the experiment to verify the method. When the derivatization process is done, the labeled glycans need to be separated from too much labeling with the SPE cartridge. On the MALDI-TOF-TOF-MS, the increased mass 120.06 Da due to the derivatization can be easily found. Then the samples with 1:1 before and after labeling are subjected to UPLC-ESI-MS-MS, the signal of the peak with H+ can be detected with an obvious improvement, proving the effecitiveness of the method, through the structural confirmation with MS/MS spectra. Then at the second step, we apply the method to the complex N-glcans mixture.The characterization of the biological medicine, including the determination of molecular mass of intact protein, and the analysis of the sequence of amino acids as well as modifications, especially with N-linked glycans, provides a possibility to identify the structure of biological drugs, especially when compared to the reference, with the precision and reliability of the results.