Comparison of partial least square, artificial neural network, and support vector regressions for real-time monitoring of CHO cell culture processes using in situ near-infrared spectroscopy.

The biopharmaceutical industry must guarantee the efficiency and biosafety of biological medicines, which are quite sensitive to cell culture process variability. Real-time monitoring procedures based on vibrational spectroscopy such as near-infrared (NIR) spectroscopy, are then emerging to support innovative strategies for retro-control of key parameters as substrates and by-product concentration. Whereas monitoring models are mainly constructed using partial least squares regression (PLSR), spectroscopic models based on artificial neural networks (ANNR) and support vector regression (SVR) are emerging with promising results. Unfortunately, analysis of their performance in cell culture monitoring has been limited. This study was then focused to assess their performance and suitability for the cell culture process challenges. PLSR had inferior values of the determination coefficient (R2 ) for all the monitored parameters (i.e., 0.85, 0.93, and 0.98, respectively for the PLSR, SVR, and ANNR models for glucose). In general, PLSR had a limited performance while models based on ANNR and SVR have been shown superior due to better management of inter-batch heterogeneity and enhanced specificity. Overall, the use of SVR and ANNR for the generation of calibration models enhanced the potential of NIR spectroscopy as a monitoring tool.

Interest of locally weighted regression to overcome nonlinear effects during in situ NIR monitoring of CHO cell culture parameters and antibody glycosylation.

Animal cell culture processes have become the standard platform to produce therapeutic proteins such as recombinant monoclonal antibodies (mAb). Since the mAb quality could be subject to significant changes depending on manufacturing process conditions, real time monitoring and control systems are required to ensure mAb specifications mainly glycosylation and patient safety. Up to now, real time monitoring glycosylation of proteins has received scarce attention. In this article, the use of near infrared (NIR) to monitor mAb glycosylation has been reported for the first time. Whereas monitoring models are mainly constructed using linear partial least squares regressions (PLSR), evidences presented in this study indicate nonlinearity relationship between in situ captured spectra and compound concentrations, compromising the PLSR performances. A novel and simple approach was proposed to fit nonlinearity using the locally weighted regression (LWR). The LWR models were found to be more appropriate for handling information contained in spectra so that real time monitoring of cultures were accurately performed. Moreover, for the first time, the LWR calibration models allowed mAb glycosylation to be monitored, in a real time manner, by using in situ NIR spectroscopy. These results represent a further step toward developing active-control feedback of animal cell processes, particularly for ensuring properties of biologics.

Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi.

A chitinolytic enzyme from Bacillus thuringiensis subsp. aizawai has been purified and its molecular mass was estimated ca. 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was able to hydrolyze chitin to chitobiosides but not carboxymethylcellulose, cellulose, pullulan, and laminarin. Optimal pH and temperature were detected at 6 and 50 degrees C, respectively. Stability, in the absence of substrate, was observed at temperatures less than 60 degrees C and pH between 5 and 8. Enzyme activity was significantly inhibited by K+ and EDTA and completely inhibited by Hg2+. Purified chitinase showed lytic activity against cell walls from six phytopathogenic fungi and inhibited the mycelial growth of both Fusarium sp. and Sclerotium rolfsii. The biocontrol efficacy of the enzyme was tested in the protection of bean seeds infested with six phytopathogenic fungi.