The stringent requirements for proving that biotherapeutic protein products are correctly folded into the active three-dimensional structure is a vital concern of the biopharma industry, as slight changes to the process environment can lead to loss of structural integrity, in turn reducing a drug’s therapeutic efficacy and invoking immune responses in patients. The recognition that biotherapeutic quality attributes of protein products are influenced by multiple factors has contributed to a series of risk-based initiatives governing their manufacture. These measures aim to safeguard product consistency, quality and purity by ensuring that the manufacturing process remains substantially the same over time. Important among these initiatives is the Food and Drug Administration’s guidance on process analytical technologies (PAT) within the pharmaceutical sector. In the case of biotherapeutic protein targets, where maintenance of the correct 3D structure is essential for drug efficacy, improved understanding of the impact of processing environments relies, in large part, on the development of robust high-throughput analytical techniques capable of effectively monitoring a given protein’s structural state at all stages during processing and formulation. PAT is increasingly being used for the monitoring of bioprocesses, and though most applications to date occur in process development (e.g. high-throughput screening of conditions) there is considerable future scope for employing PAT during manufacture (e.g. in in-process monitoring and control roles).
Circular dichroism (CD) and intrinsic fluorescence are sensitive, non-destructive biophysical techniques employed for studying changes in the solution-state conformation of proteins. CD provides an excellent means of measuring of changes in the total secondary structure of a protein, whilst absorption in the near-UV region arising from aromatic amino acids provides a good indicator of tertiary structure changes. Intrinsic protein fluorescence is a complementary technique that provides information on the state of protein structure from changes in the local environment of aromatic amino acid residues. This said, a significant challenge with using CD and fluorescence together for routine structural screening is that current instruments are not configured for use in an industrial setting, where speed and autonomy are paramount.