Capillary Electrophoresis-Mass Spectometry for the Analysis of Biopharmaceuticals

Nov 02, 2012

Developments in the fields of protein chemistry and pharmaceutical biotechnology have increased the demand for suitable analytical techniques for the characterization of intact proteins. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to be a powerful tool for this purpose because it combines high separation efficiency with mass selectivity. This article provides an overview of CE–MS method development work in our laboratory, for biopharmaceutical analysis applying noncovalent capillary coatings to prevent protein adsorption. Technological aspects with respect to the use of CE–MS interfaces and types of noncovalent capillary coatings applied are treated. Furthermore, some typical examples are highlighted to demonstrate the versatility of CE–MS for the analysis of biopharmaceuticals. These include the analysis of recombinant human growth hormone, recombinant human interferon-β-1a, recombinant human erythropoietin and protein–drug conjugates. We concluded that there is strong potential for CE–MS systems using noncovalent coated capillaries for purity and stability analysis of biopharmaceuticals.



Biopharmaceuticals have an increasingly important position in drug development and production (1). As for any drug designed for human use, biopharmaceuticals have to meet stringent quality requirements to ensure efficacy and safety. However, because of their macromolecular nature, the full characterization of biopharmaceuticals in terms of identity, content, purity, stability, conformation and function poses a great challenge (2,3). Moreover, multistep biotechnological production processes may show variability, introducing product diversity and different isoforms that can affect activity. With the appearance of biosimilars that are supposed to be copies of the innovator product (3), the demand for an effective quality assessment of therapeutic proteins has increased even further.

Next to techniques such as liquid chromatography (LC) and gel electrophoresis, capillary electrophoresis (CE) is a proven powerful separation technique for biotechnology-derived proteins (4). CE analyses can be performed under mild conditions without the need for organic solvents or very high salt concentrations. This allows the study of proteins without causing conformational changes or protein degradation during analysis. In CE, separations are based on differences in the charge-to-size ratio of the analytes. Therefore, subtle differences among proteins, such as those caused by post-translational modifications like glycosylation, deamidation, or phosphorylation, can be differentiated with CE. Mass spectrometry (MS) has developed into one of the most popular and useful detection techniques in separation science. MS is well suited for protein analysis because of its sensitivity and selectivity. Furthermore, MS detection with high mass accuracy and resolution, such as provided by time-of-flight (TOF) instruments, can considerably enhance the utility of CE by providing information about the identity of the separated compounds. Therefore, coupling CE to MS creates a powerful analytical tool for the characterization of intact proteins (5,6).

Despite its potential, the application of CE–MS for the analysis of biopharmaceuticals has been relatively limited. CE–MS methods that distinguish natural human growth hormone (hGH) from recombinant hGH (7) and reveal degradation products in hGH after heat exposure and prolonged storage (8,9) were introduced. Furthermore, the usefulness of CE–MS for the intact glycoform analysis of erythropoietin has been demonstrated (for example, references 10–12 and the references therein). During the last few years, our group has been working on the development of CE–MS methodologies for the characterization of intact proteins and biopharmaceuticals in particular. In this paper some representative data is provided showing the strength of the combination of CE with high-resolution TOF-MS for the characterization of protein drugs.