For the past two decades, the development of protein therapeutics has significantly expanded with numerous biopharmaceutical and biosimilar products entering the medicine market every year, and even more queuing in the pipeline globally. Biologics are very complex molecules and therefore extremely sensitive to minor changes in the manufacturing process, which can result in heterogeneity and affect the stability, potency and immunogenicity of the final product. Public health organisations, such as EMA (European Medicines Agency), require that biological products should be extensively tested for their similarity to the original drug (in the case of a biosimilar) as well as to products from different batches (batch-to-batch comparison). The issued guidelines focus, among other tests, on physicochemical characterisation of these molecules. The suggested analytical techniques, however, are only vaguely named in the specifications, leaving the final decision to the manufacturers. The present work focuses on the use of different combinations of analytical techniques with an aim to demonstrate similarity or dissimilarity between two or more samples. The selected instrumental techniques are characterised by their simplicity and are able to detect structural differences and microheterogeneity of the active ingredient in different samples, aggregation, degradation and post-translational modifications (PTMs). Seven studies were completed in total, each one to a different extent, and these included protein therapeutics such as insulin and monoclonal antibodies. The applied techniques served for primary (MS),* secondary (far-UV CD, FT-IR) and tertiary structure (near-UV CD, fluorescence) comparison of the examined samples. Particle size comparability and detection of aggregation was achieved with DLS, and higher-order structure comparison with 1D 1 H-NMR. Coupling of the techniques with temperature-dependent measurements enabled further comparison on the thermal stability of the samples and provided confidence in the observed (at room temperature) results. The acquired empirical experience pointed out the advantages and disadvantages of each technique compared to the rest of the techniques, possible solutions to the encountered challenges, and the cases that one technique can be used instead of another or as complementary to it. Finally, a potential SOP is suggested, advising on which biophysical techniques should be used depending on the structure of the protein that is examined and its formulation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:737694 |
| Date | January 2017 |
| Creators | Sklepari, Meropi |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/98558/ |
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