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.

A new perfusion mode of culture for WJ-MSCs expansion in a stirred and online monitored bioreactor.

As a clinical dose requires a minimum of 106 cells per kilogram of patients, it is, therefore, crucial to develop a scalable method of production of Wharton Jelly mesenchymal stem cells (WJ-MSCs) with maintained inner characteristics. Scalable expansion of WJ-MSCs on microcarriers usually found in cell culture, involves specific cell detachment using trypsin and could have harmful effects on cells. In this study, the performance of batch, fed-batch, and perfused-continuous mode of culture were compared. The batch and fed-batch modes resulted in expansion factors of 5 and 43, respectively. The perfused-continuous mode strategy consisted of the implementation of a settling tube inside the bioreactor. The diameter of the tube was calculated to maintain microcarriers colonized by cells in the bioreactor whereas empty microcarriers (responsible for potentially damaging collisions) were removed, using a continuous flow rate based on MSCs physiological requirements. Thanks to this strategy, a maximal number of 800 million cells was obtained in a 1.5 L bioreactor in 10 days. Lastly, online dielectric spectroscopy was implemented in the bioreactor and indicated that cell growth could be monitored during the culture.

Metabolomics approach based on LC-HRMS for the fast screening of iron(II)-chelating peptides in protein hydrolysates.

Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+56FeII]+ complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, ßAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+56FeII]+ complex showed a limit of detection (LOD) below 0.77 mg L-1 of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77 mg L-1 in casein hydrolysate. Graphical abstract.

In situ cell differentiation monitoring of Catharanthus roseus suspension culture processes by NIR spectroscopy.

Plant suspension culture is attracting interest as a promising platform to produce biological medicines due to the absence of virus, prions or DNA related to mammals during the production process. However, the heterogenic plant cell proliferation nature is particularly challenging for establishing industrial processes based on innovative approaches currently used, particularly in the animal cell culture industry. In this context, while Process Analytical Technology (PAT) tools have been used to monitor classical parameters such as biomass dry weight, its use in cells heterogeneity has received limited attention. Therefore, the feasibility of in situ monitoring of cell differentiation in plant cell suspensions employing NIR spectroscopy and chemometrics was investigated. Off-line measurements of cell heterogeneity in term of cell differentiation and in-line NIR spectra captured in 3 L bioreactor cultures were employed to generate calibration models. Then models were tested to estimate the population distribution of parenchyma, collenchyma and sclerenchyma cells during Catharanthus roseus suspension cultures. Results have proven in situ NIR spectroscopy as a capable PAT tool to monitor differentiated cells accurately and in real-time. These results are the starting point to follow-up PAT systems so that plant cell culture heterogeneity may be better understood and controlled in biopharmaceutical plant cell cultures.

Control of IgG glycosylation by in situ and real-time estimation of specific growth rate of CHO cells cultured in bioreactor.

The cell-specific growth rate (µ) is a critical process parameter for antibody production processes performed by animal cell cultures, as it describes the cell growth and reflects the cell physiological state. When there are changes in these parameters, which are indicated by variations of µ, the synthesis and the quality of antibodies are often affected. Therefore, it is essential to monitor and control the variations of µto assure the antibody production and achieve high product quality. In this study, a novel approach for on-line estimation of µ was developed based on the process analytical technology initiative by using an in situ dielectric spectroscopy. Critical moments, such as significant µ decreases, were successfully detected by this method, in association with changes in cell physiology as well as with an accumulation of nonglycosylated antibodies. Thus, this method was used to perform medium renewals at the appropriate time points, maintaining the values of µ close to its maximum. Using this method, we demonstrated that the physiological state of cells remained stable, the quantity and the glycosylation quality of antibodies were assured at the same time, leading to better process performances compared with the reference feed-harvest cell cultures carried out by using off-line nutrient measurements.

A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.

OBJECTIVE: to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors. RESULTS: Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 10(6) cells ml(-1), while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 10(7) cells ml(-1). Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l(-1). Considering the LDH content of viable cells measured to be 110 U per 10(9) cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R(2) ≥ 0.95) for the three types of cell populations. CONCLUSION: The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

Impact of probiotics on risk factors for cardiovascular diseases. A review.

Probiotic microorganisms have historically been used to rebalance disturbed intestinal microbiota and to diminish gastrointestinal disorders, such as diarrhea or inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis). Recent studies explore the potential for expanded uses of probiotics on medical disorders that increase the risk of developing cardiovascular diseases and diabetes, such as obesity, hypercholesterolemia, arterial hypertension, and metabolic disturbances such as hyperhomocysteinemia and oxidative stress. This review aims at summarizing the proposed molecular and cellular mechanisms involved in probiotic-host interactions and to identify the nature of the resulting beneficial effects. Specific probiotic strains can act by modulating immune response, by producing particular molecules or releasing biopeptides, and by modulating nervous system activity. To date, the majority of studies have been conducted in animal models. New investigations on the related mechanisms in humans need to be carried out to better enable targeted and effective use of the broad variety of probiotic strains.

Deciphering Rind Color Heterogeneity of Smear-Ripened Munster Cheese and Its Association with Microbiota.

Color is one of the first criteria to assess the quality of cheese. However, very limited data are available on the color heterogeneity of the rind and its relationship with microbial community structure. In this study, the color of a wide range of smear-ripened Munster cheeses from various origins was monitored during storage by photographic imaging and data analysis in the CIELAB color space using luminance, chroma, and hue angle as descriptors. Different levels of inter- and intra-cheese heterogeneity were observed. The most heterogeneous Munster cheeses were the darkest with orange-red colors. The most homogeneous were the brightest with yellow-orange. K-means clustering revealed three clusters distinguished by their color heterogeneity. Color analysis coupled with metabarcoding showed that rinds with heterogeneous color exhibited higher microbial diversity associated with important changes in their microbial community structure during storage. In addition, intra-cheese community structure fluctuations were associated with heterogeneity in rind color. The species Glutamicibacter arilaitensis and Psychrobacter nivimaris/piscatorii were found to be positively associated with the presence of undesirable brown patches. This study highlights the close relationship between the heterogeneity of the cheese rind and its microbiota.

Green strategies to control redox potential in the fermented food industry.

Lactic acid bacteria (LAB) are important microorganisms in the food industry as functional starters for the manufacture of fermented food products and as probiotics. Redox potential (Eh) is a parameter of the physicochemical environment of foods that influences key oxidation-reduction reactions involved in process performances and product quality. Eh can be modified by different methods, using redox molecules, catalytic activity of enzymes or LAB themselves, technological treatments like electroreduction or heating, and finally gases. Nowadays new applications for food manufacture must undertake green process innovation. This paper presents the strategies for Eh modification in a sustainable manner for production of LAB biomass (starters, probiotics) and fermented food products (fermented milks, cheeses and others). While the use of chemical or enzymes may be subject to controversy, the use of gases offers new opportunities, in combination with LAB. Protection against food-borne microorganisms, an increasing growth and viability of LAB, and a positive impact on food flavour are expected.

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.

Real-time monitoring of antibody glycosylation site occupancy by in situ Raman spectroscopy during bioreactor CHO cell cultures.

The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time- and labor-intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real-time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and nonglycosylated mAbs. By comparing variable importance in projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed-harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and nonglycosylated mAbs could be successfully and simultaneously estimated in real time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on-line optimization of mAb quality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:486-493, 2018.

Screening of lactic acid bacteria for reducing power using a tetrazolium salt reduction method on milk agar.

Reducing activity is a physiological property of lactic acid bacteria (LAB) of technological importance. We developed a solid medium with tetrazolium dyes enabling weakly and strongly reducing LAB to be discriminated. It was used to quantify populations in a mixed culture (spreading method) and screen strains (spot method).