Improved process analytical technology (PAT) is required in the pharmaceutical industry to enable real-time monitoring and control of bio-pharmaceutical manufacturing processes. A particular challenge is PAT in continuous processing. Among the various PAT tools, mid-infrared (Mid-IR) spectroscopy stands out for its ability to provide rapid, molecular specific non-destructive analysis of chemical compositions. Our research focuses on the monitoring of protein aggregation, a critical quality attribute in bio-pharmaceutical production of monoclonal antibodies. During the manufacturing and purification of monoclonal antibodies, the high background absorbance of aqueous solutions makes in-line transmission measurements with traditional Fourier transform infrared (FTIR) spectrometers unfeasible. With the use of a quantum cascade laser, a tuneable, high spectral power density mid-IR source, flow-through absorbance measurements of the amide I and amide II band regions of proteins are attainable. However, important issues remain: Matrix changes (e.g. buffer, pH, temperature changes) can lead to absorbance bands that overlap or distort the protein bands. This requires the use of multivariate data analysis and large calibration data sets. Due to the uncertain purity of commercial proteins and the resulting variability in the final concentration of calibration solutions, reference measurements are critical. This research aims to address the difficulties in calibrating a mid-IR spectroscopic sensor for complex samples, with a particular focus on its application in the purification of monoclonal antibodies.