High-resolution NMR is a key technology that provides critical information about protein structure and dynamics. It is perceived to be difficult to use, expensive, size limited and requires labelled molecules and time consuming. These aspects have hindered the adoption of the technique for the characterization of biotherapeutic drugs in the pharmaceutical industry. Recent advances in acquisition and analysis have changed the situation.1 We are now able to analyse intact antibodies at natural abundance. NMR is especially sensitive to changes to higher order structure at the atomic resolution, making it ideally suited for similarity assessment of biologics and biosimilars.2 NMR also allows for intact protein analysis, enabling evaluation of the structure of therapeutic drugs without modification, in conditions that are physiologically relevant. Because of its intrinsically high information content NMR has the potential to reduce the number of techniques needed to characterize therapeutic drugs.

Due to the quantitative nature of magnetic resonance and its selectivity, potency determination,3 impurity profiling4 and degradation studies (e.g. of polysorbates) are performed directly enabling fast and easy testing without the need of response factor calculations, or the method redevelopment activities required by traditional LC methods, thereby saving time and reducing costs. In these last examples size is not an issue because we either look at the small molecules or specifically at radicals. Another way to overcome the molecular size threshold is to approach the problems from a completely different angle. There have been several publications in the last 2-3 year on the use of time-domain NMR (TD-NMR) to determine aggregation5 and moisture6 in biologics. TD-NMR has the great advantage of being truly non-destructive, allowing the analysis of drugs in their container (vials and syringes) and maintaining the sterility of the product. This, together with the speed of the measurements, enables 100% testing, which is a requirement for highly potent drugs.

NMR is also being used in bio-production understanding7 and culture media screening and quality control.

Current applications of magnetic resonance (NMR, EPR, TD-NMR) for the analysis of biotherapeutic drugs will be reviewed.

1. Arbogast L., Delaglio F., Tolman J.R., J Biomol NMR, 72: 149-161 (2018)
2. Haxhom G.W., Bent O., Malmstrom J., J Pharm Sci, 108: 3029 (2019)
3. Bradley S. A., Jackson W. C., Mahoney P. P., Anal Chem. 91(3):1962-1967 (2019)
4. Skidmore K, Hewitt D, Kao Y.H., Biotechnol Prog. 28(6):1526-33 (2012)
5. Taraban M.B., Briggs K.T., Merkel P., Anal Chem. 91, 13538-13546 (2019)
6. Abraham A, Elkassabany O., Krause M.E., Ott A., Magn Reson Chem, 1-5 (2019)
7. Bradley S. A., et al, J. Am. Chem. Soc., 132 (28), 9531–9533 (2010)

What molecular attributes are responsible for the biological activity of a protein product? What changes in the molecule may affect its biological activity, stability, or immunogenicity? What structural components determine the half-life and biodistribution of the protein? When in the development of a product do we need to address these questions and what tools should be used? How are structure-function studies used in the evaluation of comparability and biosimilarity? Understanding how the structure of a molecule impacts its function and clinical profile is at the core of controlling protein manufacture and ensuring consistent clinical safety and efficacy throughout the product lifecycle. In this talk, we will discuss the regulatory expectations for evaluating structure and function in protein products, addressing what, how, when, and why to do these studies.

CE and LCMS are now being used to characterize gene therapy products and oligonucleotides under development. From this presentation you will:

• Understand how both CE and LCMS are being used to profile viral capsid proteins to gain better understanding of gene therapy product potency.
• Discover how CE is now being used to profile full and empty viral vectors used in gene therapy and profile the genome integrity of viral particles.
• Find out how CE and LCMS is being used to detect and characterize oligonucleotides including CE profiling of plasmids (used in gene therapy) and RNA products (CRISPR) and the detection and characterization of smaller oligonucleotides by LCMS.