Characterization of Intact Protein Conjugates and Biopharmaceuticals Using Ion-Exchange Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry and Top-Down Tandem Mass Spectrometry

Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or “designer” modifications such as chemical conjugation) or introduced postproduction as a result of stress (e.g., oxidation and deamidation). A combination of ion-exchange chromatography (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chemical modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymatic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural analysis, as demonstrated using stressed interferon-β as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addition to the experimental toolbox of biopharmaceutical analysis

Characterization of Small Protein Aggregates and Oligomers Using Size Exclusion Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry

Self-association of proteins is important in a variety of processes ranging from acquisition of native quaternary structure (where the association is tightly controlled and proceeds in a highly ordered fashion) to aggregation and amyloidosis. The latter is frequently accompanied (or indeed triggered) by the loss of the native structure, but a clear understanding of the complex relationship between conformational changes and protein self-association/aggregation remains elusive due to the great difficulty in characterizing these complex and frequently heterogeneous species. In this study, size exclusion chromatography (SEC) was used in combination with online detection by native electrospray ionization mass spectrometry (ESI MS) to characterize a commercial protein sample (serum albumin) that forms small aggregates. Although noncovalent dimers and trimers of this protein are readily detected by native ESI MS alone, combination of SEC and ESI MS allows a distinction to be made between the oligomers present in solution and those formed during the ESI process (artifacts of ESI MS). Additionally, native ESI MS detection allows a partial loss of conformation integrity to be detected across all albumin species present in solution. Finally, ESI MS detection allows these analyses to be carried out readily even in the presence of other abundant proteins coeluting with albumin. Native ESI MS as an online detection method for SEC also enables meaningful characterization of species representing different quaternary organization of a recombinant glycoprotein human arylsulfatase A even when their rapid interconversion prevents their separation on the SEC time scale