William S. Hancock
Penny J. Beuning, Michael P. Pollastri, Zhaohui S. Zhou
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Science. Department of Chemistry and Chemical Biology.
chemistry, analytical chemistry, antibodies, Immunoglobulin G, LC-MS, liquid chromatography-mass spectrometry
Therapeutic monoclonal antibodies (mAb) have been one of the fastest-growing areas in the biotechnological and pharmaceutical industry due to their high specificity as anticancer drugs with few side effects. Comprehensive characterization of such mAbs is critical to the biotech industry. Particularly, detection of functionality-sensitive posttranslational modifications (PTMs) and chemical modifications in recombinant monoclonal antibodies can have significant quality implications. Liquid chromatography-mass spectrometry (LC-MS) is currently the most sophisticated and powerful tool for protein characterization due to its superior resolution, sensitivity, and accuracy. This dissertation focuses on the structural characterization of antibodies (IgGs) using liquid chromatography - mass spectrometry (LC-MS) based methods.
Chapter 1 reviews the importance of mAb drugs, structure of mAbs, common chemical modifications and the current mass spectrometry methods used in their structural characterization.
In Chapter 2, the disulfide linkages present in three therapeutic mAbs, anti-HER2 mAb, anti-CD11a mAb and GLP-1 with IgG4-Fc fusion protein, were completely characterized by our LC-MS methods with both electron-transfer dissociation (ETD) and collision induced dissociation (CID) fragmentation. Scrambled disulfides in heat-stressed samples were also identified easily and confidently by this analytical method. We then compared the disulfide scrambling patterns of these three different mAbs and how the disulfide scrambling process may initiate.
In Chapter 3, a new chip-based LC-MS method for direct analysis of N-glycosylation present in intact mAbs was evaluated. Immobilized PNGase F and porous graphitized carbon (PGC) column are integrated together in this mAb-Glyco chip for on-line release of the glycans, separation and mass spectrometric analysis. For a mAb with only one glycosylation site, this automation and absence of manual sample pre-treatment simplifies the procedure and reduces analysis time significantly. The glycan distribution of anti-HER2 mAb generated from mAb-Glyco chip was compared to the results from two other chip-based MS methods: PGC chip MS for offline released glycans analysis and a C18 chip MS for glycopeptide analysis. We then evaluated day-to-day and run-to-run reproducibilities of these methods and then analyzed different types of mAbs, including innovator and biosimilar products with different formulations.
In Chapter 4, a range of LC-MS methods have been used for comprehensive characterization of the primary structure, disulfides, glycan structures, and other modifications of innovator and biosimilar anti-HER2 mAbs. The LC-MS methods include identification of glycopeptides and disulfide linkages by the combination of CID and ETD fragmentation and profiling glycans by an enzyme immobilized chip-based LC-MS approach. The extent of N-terminal pyroglutamic acid cyclization, heavy chain C-terminal lysine processing, oxidation, deamidation and isomerization were compared between innovator and biosimilar anti-HER2 mAbs.
In Chapter 5, the glycan distributions of IgGs in blood samples of patients at varying stages of AIDS were studied using two glycopeptide analysis methods and two glycan analysis methods. For glycopeptide analysis, tryptic peptides were analyzed by C18 reversed phase high-performance liquid chromatography - Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) while Lys-C digested peptides were analyzed by C18 reversed phase high-performance microfluidic chip liquid chromatography quadruple time-of-flight mass spectrometry (Q-TOF MS). The PNGase F offline released glycans were identified by microfluidic-based porous graphitized carbon (PGC) chip with Q-TOF MS or intact IgGs were directly analyzed by a mAb-Glyco chip. Glycopeptide analysis can provide site-specific information and therefore the glycan distribution of subclasses IgGs can be determined. Both glycopeptide analysis platforms provided similar IgG subclass-specific profiles and with a similar amount of measurement variation. The pros and cons of the different glycan analysis methods for the generation of total glycan profiles will be discussed in this chapter.
Wang, Yi, "Structural characterization of immunoglobulin G antibodies with LC-MS based approaches" (2012). Chemistry Dissertations. Paper 42. http://hdl.handle.net/2047/d20002572
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