Dr. Sigrid Pieters

Obtained degree of Doctor in Pharmaceutical Sciences in 2013

Near-infrared and Raman spectroscopy for the in-line monitoring of protein unfolding during freeze-drying processes

One objective of the FDA's Process Analytical Technology (PAT) initiative is to move the analysis of critical quality attributes (CQA's) from the quality control (QC) laboratory towards the manufacturing process environment (i.e. analysis based on in- , on- and at-line measurements).

Because of their non-invasive nature, their extremely simple and ultra-fast analysis, and because of the fact that they can transfer light over meters through fiber optics, Near-infrared (NIR) and Raman spectroscopy have become major players, even the prototype, in many in-line and on-line PAT applications.

In this doctoral dissertation, the potential of NIR and Raman spectroscopy has been investigated for a novel application: the automated and real-time detection of protein unfolding in freeze-dried formulations and during freeze-drying processes (in-line monitoring).

Protein unfolding during freeze-drying is a CQA as it may precede the formation of non-covalently bound aggregates in the reconstituted and thus administered drug product. Since the latter may cause immunogenicity and reduced potency it is a necessity to minimize protein unfolding during freeze-drying for delivering safe and effective lyophilized protein drug products.

The goal is to have a quality indication by evaluating the protein's conformational state at an early stage (preferably when the process is still running and still can be adjusted) without destroying the sample or interfering the process. Based on the interaction of light with sample molecules, characteristic chemical and physical information, visualized as a spectrum, could be obtained.

A first objective was to recognize the useful information about the protein's conformational status out of the complex spectra. Methods were developed to use this information in an optimal way for distinguishing between samples with and without remarkable protein unfolding. To allow using spectral (and thus multivariate) methods for evaluating future samples (e.g. from novel batches or different formulations) or to perform in-line measurements during the freeze-drying process, the influences of spectral interferences were investigated. Strategies for dealing with uninformative systematic effects were developed in order to increase the robustness of the multivariate methods and to allow in-line use during the freeze-drying process.

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  • Hansen, L.; Pieters, S.; Montenez, J.P.; Daoussi, R.; Vander Heyden, Y.; Vervaet, C.; Remon, J.P.; De Beer, T. (2013). Near-infrared spectroscopic evaluation of lyophilized viral vaccine formulations. Biotechnology Progress,29, 1573-1586.
  • Pieters, S.; Vander Heyden, Y.; Roger, J.M.; D'Hondt, M.; Hansen, L.; Palagos, B.; De Spiegeleer, B.; Remon, J.P.; Vervaet, C.; De Beer, T. (2013). Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations. European Journal of Pharmaceutics and Biopharmaceutics, 85, 263-271.
  • Pieters, S.; Saeys, W.; Van Den Kerkhof, T.; Goodarzi, M.; Hellings, M.; De Beer, T.; Vander Heyden, Y. (2013). Robust calibrations on reduced sample sets for API content prediction in tablets: Definition of a cost-effective NIR model development strategy. Analytica Chimica Acta, 761, 62-70.
  • Pieters, S.; De Beer, T.; Vander Heyden, Y. (2012). Near-infrared and Raman spectroscopy: potential tools for monitoring of protein conformational instability during freeze-drying processes. American Pharmaceutical Review, 15, 66-73.
  • Pieters, S.; De Beer, T.; Kasper, J.C.; Boulpaep, D.; Friess, W.; Remon, J.P.; Vervaet, C.; Waszkiewics, O.; Goodarzi, M.; Tistaert, C.; Vander Heyden, Y. (2012). Near-infrared spectroscopy for in-line probing protein denaturation and lyoprotection during freeze-drying. Analytical Chemistry, 84, 947-955.