Discrimination among IgG1-κ monoclonal antibodies produced by two cell lines using charge state distributions in nanoESI-TOF mass spectra

Abstract

Charge state distributions (CSDs) of proteins in nanoESI mass spectra are affected by the instrumental settings and experimental conditions, in addition to the conformations of the proteins in the analyzed solutions. In the presented study, instrumental and experimental parameters-the desolvation gas flow rate, temperature, pH, buffer (ammonium acetate), and organic modifier (methanol) concentrations-were optimized according to a reduced central composite face experimental design to maximize the separation of CSDs of monoclonal IgG1-antibodies produced by two production systems (CHO and GS-NS0 cell lines). Principal component analysis and Fisher linear discriminant analysis were then used to reduce the dimensions of the acquired dataset and quantify the separation of the protein classes, respectively. The results show that the IgG1-molecules produced by the two production systems can be clearly distinguished using the described approach, which could be readily applied to other proteins and production systems. E lectrospray ionization mass spectrometry (ESI-MS) is a highly suitable technique for structural analysis of various macromolecules, partly because of the possibility of analyzing multiply charged ions from biological molecules, e.g., proteins in solution [1,2]. Further advantages for such analyses are offered by nanoESI-MS [3] (compared with conventional ESI), including reductions in sample consumption, greater tolerance of nonvolatile salts, and the softness of the technique. However, although nanoESI is a preferable ionization method for structural MS analyses of biological macromolecules, nanoESI has been shown to be highly dependent upon the geometry of the nanoflow needle; thus some needle-to-needle irreproducibility is to be expected [4].Charge-state distributions (CSD) of biological macromolecules in ESI-MS are being increasingly employed for exploring their conformations in solutions [4,5]. Changes in protein conformation can result in changes in the CSD in an ESI mass spectrum [6,7]. The mechanism whereby (nearly) desolvated ions are generated from a charged droplet is not completely understood [8]. Proteins acquire more charges in solutions that promote unfolding than the same proteins in solutions that promote folded conformations [7], but it should be noted that other factors than protein compactness may also contribute to differences in CSD patterns [9 -11]. For instance, in addition to altering the conformation of proteins, changes in the solvent also affect the ionization process, i.e., proton-transfer reactions. Thus, the solvent and the operating conditions have different conformational effects on proteins with differing stability during the electrospray process. Hence, understanding the influence of experimental conditions on protein CSDs is important to extract the structural information they may contain.In this study, the effects of selected instrumental and experimental parameters on the CSDs of IgG1-monoclonal antibodies produced by two production systems [Chinese hamster ovary (CHO)...

Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars

“…In IMS, information about protein conformation is generated by probing the collisional cross-section of the molecule in the gas phase 88 . The utility of this information, however, is dependent on preserving the native state or important attributes of a protein’s native-like structure during ionization and transition into the gas phase; this is something that is now well understood 88,89 , especially from work on native MS of proteins 90 and protein complexes 91 . IMS can be used to characterize, among other things, the effects of pegylation of protein therapeutics 92 , potential lead antibody products 93 and other aspects of antibody parameters 31 , as well as diagnose the presence of aggregates 94 (FIG.…”

“…Such possibilities include the coupling of ion exchange chromatography (IEC) separation to MS (using an MS-friendly buffer system) to conduct IEC-native or native-like MS 64,90,91,94 . Here, charge-state distributions can be utilized to extract information on the various separated drug variants to assess their conformation and aggregation state.…”

Conformation and dynamics of biopharmaceuticals: Transition of mass spectrometry-based tools from academe to industry

“…These limitations notwithstanding, probing non-covalent associations by direct ESI MS is increasingly used in the drug discovery process (31), including optimization of protein drugs (32). Charge state distribution analysis of protein ions in ESI MS is also beginning to enjoy recognition as a useful tool for comparability studies of related biopharmaceutical products (33). …”

Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

“…Nevertheless, there is growing evidence, at least in the case of stressed protein therapeutics, that detection of unfolding events by direct ESI MS is not affected by the solvent exchange step (Bobst et al 2008; Bobst et al 2010). Charge state distribution analysis of mAb ions produced by ESI MS has also been used in comparability studies and allowed molecules produced by the two production systems to be distinguished (Zamani et al 2009). …”