Advancing QCL-IR spectroscopy of proteins for DSP monitoring

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 parallel and independent 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 a model protein. 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.

This project was focused on laser-based infrared spectroscopy for process analytics in biotechnological production - more precisely in downstream processing. A fundamental challenge in downstream processing is to generate and/or establish the correct folding of bio-technologically produced proteins. State-of-the-art process analytical tools are currently not able to determine protein secondary structure rapidly and continuously. While conventional infrared spectroscopy is able to distinguish and quantify protein secondary structures it is generally neither sensitive nor robust enough for application in downstream processing. If the use of novel laser light sources can be leveraged to improve the performance of mid-IR laser spectroscopy sufficiently faster and more efficient biotechnological production can be enabled. For this purpose, this project leveraged the specific properties of infrared lasers: their high spectral power density and their coherence. Furthermore, a special focus was on methods to convert spectroscopic data into chemical information i.e. chemometrics. Specifically, methods for chemometric data evaluation using multivariate curve resolution - alternating least squares (MCR-ALS) were extended and improved. Combining improved infrared spectroscopy and data evaluation, within this project inline secondary structure determination in a key unit operation of downstream processing - product purification by preparative liquid chromatography - was demonstrated. Furthermore, leveraging improved data processing protein stability and denaturation could be studied even in presence of strongly absorbing media components. A fundamental improvement of infrared laser spectroscopy was the development of a balanced detection method that suppressed laser noise to a large degree. Infrared dispersion spectroscopy was also researched during this project. This method leverages the laser coherence to achieve a large dynamic range.