The aim of this project is to advance laser-based IR spectroscopy methods for analysis of proteins as well as to apply and establish IR spectroscopy as a monitoring and quality control tool for studying downstream bioprocesses.
Unique optical properties of quantum cascade laser (QCL) light sources enable novel approaches in IR spectroscopy. Due to the coherent nature of the QCL radiation, a laser-based Mach-Zehnder interferometer (MZI) can be implemented for simultaneous acquisition of absorption and refraction index spectra of protein samples. Within the project, the robustness and limit of detection of the measurements will be improved by this new approach of spectra recording. These advancements allow expanding the capabilities of IR spectroscopy in qualitative and quantitative analysis of proteins.
Subsequently, enabled by this progress in instrument design and performance, the developed MZI is employed to investigate individual bioprocess steps in the recombinant production of heme-containing chlorite dismutase. In downstream bioprocessing, low levels of protein concentration have been prohibitive so far for employing IR spectroscopy for process monitoring. QCL-IR spectroscopy will be introduced as tool for structure-based product analytics at significant points along downstream bioprocessing (IB analytics, refolding kinetics, downstream chain analytics and heme incorporation kinetics) and utilized for characterization and quality control of the targeted unit operations. Changes in structure and activity of the intermediate products and the final protein are correlated and effects of systematically varied process parameters will be investigated.
The successful realization of the project will establish a new QCL-IR toolbox for downstream bioprocess monitoring. The obtained data will be used for gathering process understanding to identify relationships between process parameters and product quality attributes as well as product quantity. Hybrid models, in which mechanistic and data-driven hypotheses are merged, will be formulated to gain new insights and provide model-based control for optimization of downstream unit operations.
