William S. Hancock
Paul Vouros, David A. Forsyth, Zhaohui S. Zhou
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Arts and Sciences. Department of Chemistry and Chemical Biology.
Mass spectrometry, Protein drugs, Proteomics
Protein drugs, Pharmaceutical biotechnology
Biochemistry | Medicinal and Pharmaceutical Chemistry
Protein drugs in biotechnology manufacturing are often complex because of the complexity of the manufacturing procedure and the chemical complexity of protein. During the past several years, there has been increasing interest in the development of follow-on protein drugs in light of advances in manufacturing technology, process control, and protein characterization. Biopharmaceutical and follow-on protein analysis is playing a critical rule in aiding the regulation of generic protein drugs. This dissertation focuses on the characterization and comparison of different recombinant therapeutic proteins by the application of liquid chromatography coupled online with tandem mass spectrometry (LC-MS) technology. Method development for the characterization of disulfide-linked peptides in therapeutic proteins is also presented. Chapter 1 reviews the development of biopharmaceutical and follow-on protein drugs and the technologies being used in the characterization of recombinant therapeutic proteins which include the LC-MS analysis and electron transfer dissociation (ETD)/collision induced dissociation (CID) methodology. Chapter 2 described a detailed characterization of recombinant human growth hormone that included the identification of the entire sequence with disulfide linkages as well as subtle modifications by a sensitive LC-MS approach using the accurate peptide mass (Fourier Transform Ion Cyclotron Resonance(FTICR) MS) and sequence assignment (MS/MS measurement). The extent of oxidation, deamidation, and chain cleavages were measured by the ratio of peak areas of the nonmodified peptide vs. the sum of peak area of the nonmodified and modified peptides in the same LC-MS analysis. The subtle but distinct differences were found in the recombinant human growth hormone from the three manufacturers (the follow-on, counterfeit, and the original innovator products). In chapter 3, TNK-Tissue Plasminogen Activator (TNK-tPA) samples from the innovator and follow-on manufacturers were characterized and compared. All tryptic peptides including N-terminal, C-terminal and mutated peptides as well as the disulfidelinked peptides were identified, with the demonstration of the same primary sequence and disulfide linkages between the innovator and follow-on products. The three N-linked and one O-linked fucose glycosylation sites were identified. The two N-linked (N103 and N448) and one O-linked fucose (T61) sites were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially glycosylated and was shown to have a ~2.5x difference between the innovator (60% occupancy) and follow-on (25% occupancy) products. The cleavage site for the conversion of the zymogen form to an active enzyme was identified as being between R275 and I276, with a cleavage of 40% for the innovator and 10% for the follow-on products. Chapter 4 developed an online LC-MS strategy combining collision-induced dissociation (CID-MS2), electron-transfer dissociation (ETD-MS2), and CID of an isolated product ion derived from ETD (MS3) that has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS3 step. The online LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a therapeutic monoclonal antibody, and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS3 step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance not only provided facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The online LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins. Chapter 5 described the identification of the unpaired cysteine status and mapping of the 17 disulfides of recombinant tissue plasminogen activator (rt-PA) using LC-MS with ETD/CID. The analysis was conducted using a multifragmentation approach consisting of ETD and CID, in combination with a multienzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide linked peptides, even those containing N- or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CIDMS3 to target partially disulfide-dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress.
Jiang, Haitao, "Mass spectrometric analysis of biopharmaceutical and follow-on protein drugs" (2010). Chemistry Dissertations. Paper 18. http://hdl.handle.net/2047/d20000242
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