Validation of a MALDI Intact Cell Mass Spectrometry Workflow for Routine Application in Monitoring Industrial E. coli Fermentations

Today, matrix‐assisted laser desorption/ionization‐intact cell mass spectrometry (MALDI‐ICMS) is used in clinical environments for rapid pathogen identification, referred to as MALDI biotyping. This method relies on the generation of characteristic mass spectrometric profiles of intact microorganisms. Single‐celled organisms are often used for pharmaceutical production, while this allows for the production of complex products, close monitoring and control of these processes is essential. In this study, we propose the use of MALDI‐ICMS as a potential at‐line approach for fast monitoring of the progress of a biotechnologically relevant fermentation process employing E. coli as a host. We demonstrate the reproducibility of the method, thereby indicating its potential to predict the progress of antibody fragment production in E. coli in an industrial setting. We evaluated various aspects of sample preparation, including the choice of matrix and solvent concentration, sample loading, and sample‐to‐matrix ratios. Our workflow allowed monitoring of over 100 signals across a 32‐h span of E. coli fermentation. To control instrumental variability, we performed eight technical replicates and achieved excellent repeatability, with 9% median relative standard deviation of peak intensities. Principal component analysis revealed strong time‐related differences of the mass spectra, allowing to differentiate the data based on fermentation phases. Furthermore, to assess direct monitoring of product expression, we adapted acquisition parameters to enable product detection via MALDI‐ICMS. The presented MALDI‐ICMS workflow allowed reliable monitoring of cellular changes during fermentation, thereby expanding the applications of MALDI‐ICMS to process analytical technologies (PAT).