In situ and Real-time Monitoring of Multiple Biochemicals During Microbial Process Development Utilizing Raman Spectroscopy

This feasibility study was successful at showing capability of measuring multiple bioprocess biochemicals in situ and in real-time using the Kaiser Raman equipment. Specifically it was undertaken to quantify, in real-time, the concentrations of key biochemicals during a Pichia pastoris fermentation process via the use of in situ Raman spectroscopy. From a process development perspective, monitoring the changes of key biochemicals over time, utilizing process analytical technology (PAT), such as in situ Raman spectroscopy, increases (i) process knowledge, (ii) provides real-time data to evaluate the process “state of control” to make informed process decisions, and (iii) use the biochemical predicted concentrations to be inputs of the automation to operate under normal manufacturing conditions.  This study investigated how changes to the fermentation process – including changes to the cell line, media components and pH set-point – impacted the quantitative model predictions of key biochemicals.  In total, Raman spectra collected from 14 different fermentation conditions were used for both modeling and validation.  The multivariate modeling approach entailed selecting regions of the spectra sensitive to a specific biochemistry as-well-as taking into consideration regions that displayed matrix interactions with other molecules that affected concentration.  Calibration models were developed using partial least squares (PLS) regression employing empirical data (i.e. offline assay concentration values acquired at various time points throughout each fermentation batch) of methanol, glycerol and sorbitol biochemistries.  To confirm the sensitivity of spectral regions, as well as to expand the concentration range and refine the quantitative models, spiking events were also performed.