The higher order (secondary, tertiary and quaternary) structure of a protein guides its function and is a key contributing factor to the quality, safety and efficacy of biopharmaceutical products. It is therefore important to monitor a biotherapeutic’s higher order structure (HOS) in order that any change in conformation can be measured and understood. Changes in HOS are known, for example, to lead to increases in aggregation (creating species from oligomers to visible particles) which give rise to issues with immunogenicity and activity.

Most commonly, analysis of HOS is performed during early and late characterization and also in comparability studies as stipulated by ICH Q5(E). One development area in which assessment of HOS is proving particularly critical is that of biosimilar molecules, for which different processes may lead to subtle differences in expressed protein structure. Often, structural analysis methods are able to indicate a change long before it is detected by other means.

Both scientists and regulators are increasingly aware of the important role that HOS plays in defining a biopharmaceutical’s critical quality attributes (CQAs): the first hurdle in implementing a Quality by Design (QbD) approach to development and manufacturing. The earlier that information regarding HOS is available, the more useful this knowledge is in assisting formulation, stability assessment, and process development. In line with QbD requirements, analysis of HOS is increasingly becoming a regulatory expectation.

Malvern’s technologies can provide useful information for HOS monitoring, with our most relevant solutions being the MicroCal DSC range and the Zetasizer Helix:

Malvern’s MicroCal Differential Scanning Calorimeters (DSCs) measure the heat released by a protein unfolding as temperature is increased. The thermal profiles produced are very sensitive to even subtle changes in the higher order structure and stability of proteins and biotherapeutics, making the technique ideal for monitoring the integrity of the biologic in various formulations and for process optimization and development.

The Zetasizer Helix builds upon the strength of Malvern’s flagship Zetasizer range of products with the addition of Raman spectroscopy to enable DLS measurements to be carried out simultaneously with a highly sensitive spectroscopic determination of a protein’s secondary and tertiary structure.The technique can be used under standard formulation conditions to elucidate the molecular mechanisms that drive many oligomerization, aggregation and agglomeration events or to provide complementary and supporting thermodynamic data to DSC.