Capillary electrophoresis – mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals

With efficient methodologies available in biotechnology today, increasing numbers of recombinantly manufactured pharmaceutical peptides and proteins are being commercialized. The assessment of biopharmaceutical quality in terms of identity, content and purity is an important issue during manufacturing. The biotechnological production process may show variability, which can introduce product diversity, isoforms and closely-related degradation products. Clearly, there is an increasing demand for suitable analytical methods that allow not only protein and peptide identification, but also the separation and quantification of impurities and possible degradation products Capillary electrophoresis-mass spectrometry (CE-MS) provides the high separation efficiency and mass-selective detection that can be very useful for biopharmaceutical product characterization. Many protein modifications, like glycosylation and deamidation, may involve changes of net charge of a protein and, thus, also of its electrophoretic mobility. MS detection of high mass accuracy and resolution, such as provided by time-of-flight (TOF) instruments can yield accurate information on the molecular weight of analyzed proteins species. In this thesis, the potential of CE-TOF-MS for the analysis of biopharmaceuticals has been explored. Emphasis was on development of CE methods for basic proteins, paying particular attention to the prevention of protein adsorption, which deteriorates the separation efficiency. A positively charged Polybrene-dextran sulfate-Polybrene (PB-DS-PB) coating was applied for this purpose. These coatings were prepared by successive flushes with solutions of the charged polymers, leading to formation of electrostatically adsorbed layers. The performance and stability of the triple-layer coating was evaluated for basic proteins analyzed at medium and low pH. The suitability of the triple-layer coating for CE-TOF-MS was studied, paying attention to compatibility. CE-MS coupling was achieved via a sheath-liquid interface, and optimum interfacing conditions were investigated. A disadvantage of this type of interfacing is that the sheath liquid dilutes the CE effluent leading to reduced detection sensitivity. In order to avoid this drawback, the performance of a prototype sheathless interface for intact protein analysis by CE-MS was investigated and compared to sheath-liquid CE-MS. The potential of the developed CE-TOF-MS systems was evaluated by analyzing various pharmaceutical proteins, addressing issues such as purity, stability, heterogeneity, and product composition.