In Situ Near-Infrared (NIR) versus High-Throughput Mid-Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

Abstract

The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coli cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R(2)) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

Molecular fingerprint of human gastric cell line infected by Helicobacter pylori

“…Therefore FTIRspectroscopy captured the specific biomolecular signature of infected AGS cells due to different H. pylori strains. This high sensitivity of FTIR spectroscopy has also been observed in other applications, as to monitor microbial and mammalian cell cultures, bacteria typing and sub-typing, detection of cell cycle events and the mechanisms of cell death, monitoring the differentiation of stem cells and diseases diagnosis and research [9][10][11][12][13][14].…”

Metabolic profiling of recombinant Escherichia coli cultivations based on high‐throughput FT‐MIR spectroscopic analysis

“…The model system chosen is based on the plasmid production, provided by its current relevance for DNA vaccines and gene therapy. The E. coli DH5α strain producing pVAx‐LacZ was chosen based on previous works on the development of monitoring techniques of plasmid bioproduction by NIR and MIR spectroscopies . It will be highly useful to complement these works, based on PLS regression models, with the most used qualitative multivariate data analysis (the PCA) and the direct extraction of biochemical and metabolic information from the IR spectra.…”

“…From this suspension 25 µL triplicate samples were placed on IR‐transparent Si microtiter plates with 96 wells (Bruker Optics, Germany) and subsequently dehydrated in a vacuum desiccator (ME2, Vaccubrand, Germany). A FT‐MIR spectrum per well was recorded in transmission mode by a HTS‐XT associated to Vertex‐70 spectrometer (Bruker Optics), using a spectral resolution of 2 cm −1 and 40 scans per sample . The time between all steps before IR spectral acquisition was minimized so that biochemical and metabolic changes were minimized too.…”

“…An advantage of FT‐MIR spectroscopy using the traditional transmission mode is the possibility of the at‐line spectral acquisition in high‐throughput mode, using plates with multi‐microwells that enable the simultaneous analysis of hundred micro‐samples, only requiring dehydration of the culture broth before analysis. More information regarding MIR and NIR spectroscopic capabilities can be found in Sales et al…”

“…To evaluate the monitoring system, and considering the relevance of using glucose as the main carbon‐source to promote bacterial growth, and glycerol to minimize the production of acetate, two E. coli cultures were conducted with different mixtures of glucose and glycerol and different cultivation strategies (batch and fed‐batch). In a previous work, this expression system was used to develop PLS models to predict the critical variables of the bioprocess along the culture (using the NIR and MIR regions of the spectra), namely de E. coli growth, plasmid production, C‐sources consumption and acetate production and consumption . In this work, a PCA of the spectral data was first performed as a preliminary evaluation of the FT‐MIR spectroscopy's ability to identify relationships between spectral data and main growth phases.…”