Rohin Mhatre

Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon beta-1a (IFN-beta-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-beta1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-beta1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-beta1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.

Proteomic Profiling of a High-Producing Chinese Hamster Ovary Cell Culture

The productivity of mammalian cell culture expression systems is critically important to the production of biopharmaceuticals. In this study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-X(L) gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomarker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene that is known to have growth regulating properties in human cells. A total of 392 proteins were identified in this study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were downregulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-X(L). The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures.

Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.