An important aspect in analytical assessment of pharmaceutical proteins is the comprehensive characterization of their higher order structure (HOS). This project explores the use of 2D NMR spectroscopy for in-depth analyses of HOS of bioactive proteins. It is well-established that the biological functions of proteins depend on their specific structures. However, the quality of bioactive proteins is traditionally assessed indirectly by cell-based potency assays. These assays usually take several days, are laborious, and have considerable variability. In addition, development of suitable assays for oral formulations is facing severe challenges due to excipient interference. We hypothesize that 2D NMR will provide more detailed information on the HOS of pharmaceutical proteins and will be more sensitive towards changes occurring during production including oxidation, degradation, and chemical modifications such as acylation compared to the cell-based assays. However, the feasibility and robustness of 2D NMR on proteins at natural abundance is not well described and little has been published on pharmaceutical proteins. Furthermore, the correlation between the cell-based potency assays and 2D NMR data is so far unexplored and will therefore be investigated as part of the project.
The need for a rapid, precise, sensitive, and robust HOS assay has increased since the regulatory agencies are now approving “biosimilar” pharmaceutical proteins based on HOS similarities, e.g. Zarxio. The most sensitive and detailed method for measuring HOS in proteins is unquestionable 2D NMR spectroscopy. Furthermore, solution state NMR analysis has the advantage of not requiring sample manipulation, i.e. data can be acquired directly on the drug substance or the formulated protein. Hence, the detailed HOS characterization during drug product development will lead to a better control and differentiation of proteins produced by different processes or sources.
A part of our work is focused on showing that we can measure a correlation between the bioactivity and the NMR data. This will demonstrate that NMR can be used to ensure that the structure and thereby the activity of a protein is intact. This is done by measuring the bioactivity and NMR data on intact samples as well as samples that have been stressed under extreme conditions. Combined with the variance on sample handling, the uncertainty on the NMR measurements, and batch-to-batch differences we expect to be able to set a limit on how much a given sample can deviate from an intact standard.
Dr. Joan Malmstrøm
CMC Analytical Support, R&D, Novo Nordisk A/S, Måløv, Denmark