Model Transferability and Reduced Experimental Burden in Cell Culture Process Development Facilitated by Hybrid Modeling and Intensified Design of Experiments

Reliable process development is accompanied by intense experimental effort. The utilization of an intensified design of experiments (iDoE) (intra-experimental critical process parameter (CPP) shifts combined) with hybrid modeling potentially reduces process development burden. The iDoE can provide more process response information in less overall process time, whereas hybrid modeling serves as a commodity to describe this behavior the best way. Therefore, a combination of both approaches appears beneficial for faster design screening and is especially of interest at larger scales where the costs per experiment rise significantly. Ideally, profound process knowledge is gathered at a small scale and only complemented with few validation experiments on a larger scale, saving valuable resources. In this work, the transferability of hybrid modeling for Chinese hamster ovary cell bioprocess development along process scales was investigated. A two-dimensional DoE was fully characterized in shake flask duplicates (300 ml), containing three different levels for the cultivation temperature and the glucose concentration in the feed. Based on these data, a hybrid model was developed, and its performance was assessed by estimating the viable cell concentration and product titer in 15 L bioprocesses with the same DoE settings. To challenge the modeling approach, 15 L bioprocesses also comprised iDoE runs with intra-experimental CPP shifts, impacting specific cell rates such as growth, consumption, and formation. Subsequently, the applicability of the iDoE cultivations to estimate static cultivations was also investigated. The shaker-scale hybrid model proved suitable for application to a 15 L scale (1:50), estimating the viable cell concentration and the product titer with an NRMSE of 10.92% and 17.79%, respectively. Additionally, the iDoE hybrid model performed comparably, displaying NRMSE values of 13.75% and 21.13%. The low errors when transferring the models from shaker to reactor and between the DoE and the iDoE approach highlight the suitability of hybrid modeling for mammalian cell culture bioprocess development and the potential of iDoE to accelerate process characterization and to improve process understanding.

Chitosan and Lemon Extract Applied during Giuncata Cheese Production to Improve the Microbiological Stability

In this study, lemon extract and chitosan were used as antimicrobial agents during Giuncata cheese production in order to assess whether the natural compounds would improve the cheese’s microbial quality. In particular, the viable cell concentration of the main spoilage microbial growth (Pseudomonas spp. and total coliforms) was monitored during refrigerated storage at 4 °C. A central composite design (CCD) was adopted to highlight a possible synergic effect of the two selected compounds. The results showed that a decrease in the cell growth rate of the monitored spoilage microorganisms was observed for all cheese samples added with active agents, when compared with the control cheese. Despite the recorded antimicrobial activity, an antagonist effect was detected when the two compounds were combined at the highest concentrations. In fact, the best performance was obtained when the lemon and the chitosan were used individually at concentrations of 500 and 60 ppm, respectively.

Microbes in deionized water: Implications for maintenance of laboratory water production system

Microbes, with their vast metabolic capabilities and great adaptability, occupy almost every conceivable ecological niche on Earth. Thus, could they survive in the oligotrophic (i.e., nutrient poor) deionized (DI) water that we use for experiments? Observations of white cauliflower-like lumps and black specks in salt solutions after months of storage in plastic bottles prompted the inquisition concerning the origin and nature of the “contaminants”. Hypothesizing that the “contaminants” may be microbes from DI water, a series of growth experiments was conducted to detect and profile the diversity of microbes in fresh DI water, produced on a just-in-time basis by a filter cum ion exchange system with tap water as feed. While microbes could also be present on the surfaces and headspace of the unsterilized polyethylene bottles, investigating whether microbes are present in freshly produced DI water provides a more stringent performance test of the production system. Inoculation of DI water on R2A agar followed by multi-day aerobic cultivation revealed the presence of a wide variety of microbes (total viable cell concentration of ~103 colony forming units (CFU) per mL) with differing pigmentations, growth rates, as well as colony sizes and morphologies. Additionally, greater abundance and diversity of microorganisms was recovered at 30 oC compared to 25 and 37 oC; most probably due to adaptation of microbes to tropical ambient water temperatures of 25 to 30 oC. Comparative experiments with tap water as inoculum recovered a significantly smaller number and diversity of microorganisms; thus, suggesting that monochloramine residual disinfectant in tap water was effective in inhibiting cell viability. In contrast, possible removal of monochloramine by adsorption onto ion exchange resins (and thus, alleviation of a source of environmental stress) might explain the observed greater diversity and abundance of viable microbes in DI water. Collectively, this study confirmed the presence of microbes in fresh DI water, and suggested a possible source of the “contaminants” in prepared salt solutions. Propensity of microbes in forming biofilm on various surfaces suggested that intermittent flow in just-in-time DI water production provided opportunities for cell attachment and biofilm formation during water stagnation, and subsequent dislodgement and resuspension of cells upon water flow. Thus, regular maintenance and cleaning of the production system should help reduce DI water’s microorganism load. In addition, simple and low cost culture experiments on solid medium can provide a qualitative and semi-quantitative estimate of microbial diversity and viable cell concentration in DI water, respectively. The above, together with regular monitoring of water resistivity or conductivity, comprise a trio of tests useful for detecting possible contamination, or deterioration of DI water’s chemical and microbiological quality.

Microbes in deionized water: Implications for maintenance of laboratory water production system

Microbes, with their vast metabolic capabilities and great adaptability, occupy almost every conceivable ecological niche on Earth – thus, could they survive in the oligotrophic (i.e., nutrient poor) deionized (DI) water that we use for experiments? Observations of white cauliflower-like lumps and black specks in salt solutions after months of storage in plastic bottles prompted the inquisition concerning the origin and nature of the “contaminants”. Hypothesizing that the “contaminants” may be microbes from DI water, a series of growth experiments was conducted to detect and profile the diversity of microbes in fresh DI water - produced on a just-in-time basis by a filter cum ion exchange system with tap water as feed. While microbes could also be present on the surfaces and headspace of the unsterilized polyethylene bottles, investigating whether microbes are present in freshly produced DI water provides a more stringent performance test of the production system. Inoculation of DI water on R2A agar followed by multi-day aerobic cultivation revealed the presence of a wide variety of microbes (total viable cell concentration of ~103 colony forming units (CFU) per mL) with differing pigmentations, growth rates as well as colony sizes and morphologies. In addition, greater abundance and diversity of microorganisms was recovered at 30 oC compared to 25 and 37 oC; most probably due to adaptation of microbes to tropical ambient water temperatures of 25 to 30 oC. Comparative experiments with tap water as inoculum recovered a significantly smaller number and diversity of microorganisms; thus, suggesting that monochloramine residual disinfectant in tap water was effective in inhibiting cell viability. In contrast, possible removal of monochloramine by adsorption onto ion exchange resins – and thus, alleviation of a source of environment stress - might explain the observed greater diversity and abundance of viable microbes in DI water. Collectively, this study confirmed the presence of microbes in fresh DI water – and suggested a possible source of the “contaminants” in prepared salt solutions. Propensity of microbes in forming biofilm on various surfaces suggested that intermittent flow in just-in-time DI water production provided opportunities for cell attachment and biofilm formation during water stagnation, and subsequent dislodgement and resuspension of cells upon water flow. Thus, regular maintenance and cleaning of the production system should help reduce DI water’s microorganism load. In addition, simple and low cost culture experiments on solid medium can provide a qualitative and semi-quantitative estimate of microbe diversity and viable cell concentration in DI water, respectively. The above, together with regular monitoring of water resistivity or conductivity, comprise a trio of tests useful for detecting possible contamination, or deterioration of DI water’s chemical and microbiological quality.

Microbes in deionized water: Implications for maintenance of laboratory water production system

Microbes, with their diverse metabolic capabilities and great adaptability, occupy almost every conceivable ecological niche on Earth – thus, could they survive in the oligotrophic (i.e., nutrient-poor) deionized (DI) water that we use for our experiments? Observations of white cauliflower-like lumps and black specks in salt solutions after months of storage in plastic bottles prompted the inquisition concerning the origin and nature of the “contaminants”. Hypothesizing that the “contaminants” may be microbes from DI water, a series of growth experiments was conducted to detect and profile the microbial diversity in fresh DI water - produced on a just-in-time basis by a filter-cum-ion-exchange system with tap water as feed. While microbes could also be present on the surfaces and headspace of the unsterilized polyethylene bottles, investigating whether microbes are present in freshly produced DI water provides a more stringent performance test of the production system. Inoculation of DI water on R2A agar followed by multi-day aerobic cultivation revealed the presence of a wide variety of microbes (total viable cell concentration of ~103 colony forming units (CFU) per mL) with differing pigmentations, growth rates as well as colony sizes and morphologies. Additionally, greater abundance and diversity of microbes was recovered at 30 oC relative to 25 and 37 oC; most probably due to adaptation of microbes to tropical ambient water temperatures of 25 to 30 oC. Comparative experiments with tap water as inoculum recovered a significantly smaller number and diversity of microbes; thereby, suggesting that monochloramine residual disinfectant in tap water was effective in inhibiting cell viability. In contrast, possible removal of monochloramine by adsorption onto ion-exchange resins – and thus, alleviation of a source of environmental stress - might explain the observed greater diversity and abundance of viable microbes in DI water. Collectively, this study confirmed the presence of microbes in fresh DI water – and suggested a possible source of the “contaminants” in prepared salt solutions. Propensity of microbes for forming biofilm on various surfaces suggested that intermittent flow in just-in-time DI water production provided opportunities for cell attachment and biofilm formation in the system during water stagnation, and subsequent dislodgement and resuspension of cells upon water flow. Thus, regular maintenance and cleaning of the production system should help reduce DI water’s microbial load. Additionally, simple and low-cost culture experiments on agar medium can provide a qualitative and semi-quantitative estimate of microbial diversity and viable cell concentration in DI water, respectively, and along with regular monitoring of water resistivity or conductivity, comprise a trio of tests useful for detecting possible contamination, or deterioration of DI water’s chemical and microbiological quality.

Microbes in deionized water: Implications for maintenance of laboratory water production system

Microbes, with their diverse metabolic capabilities and great adaptability, occupy almost every conceivable ecological niche on Earth – thus, could they survive in the oligotrophic (i.e., nutrient-poor) deionized (DI) water that we use for our experiments? Observations of white cauliflower-like lumps and black specks in salt solutions after months of storage in plastic bottles prompted the inquisition concerning the origin and nature of the “contaminants.” Hypothesising that the “contaminants” may be microbes from DI water, a series of growth experiments was conducted to detect and profile the microbial diversity in fresh DI water - produced on a just-in-time basis by a filter-cum-ion-exchange system with tap water as feed. While microbes could also be present on the surfaces and headspace of the unsterilized polyethylene bottles, investigating whether microbes are present in freshly produced DI water provides a more stringent performance test of the production system. Inoculation of DI water on R2A agar followed by multi-day aerobic cultivation revealed the presence of a wide variety of microbes (total viable cell concentration of ~103 colony forming units (CFU) per mL) with differing pigmentations, growth rates as well as colony sizes and morphologies. Additionally, greater abundance and diversity of microbes was recovered at 30 oC relative to 25 and 37 oC; most probably due to adaptation of microbes to tropical ambient water temperatures of 25 to 30 oC. Comparative experiments with tap water as inoculum recovered a significantly smaller number and diversity of microbes; thereby, suggesting that monochloramine residual disinfectant in tap water was effective in inhibiting cell viability. In contrast, possible removal of monochloramine by adsorption onto ion-exchange resins – and thus, alleviation of a source of environmental stress - might explain the observed greater diversity and abundance of viable microbes in DI water. Collectively, this study confirmed the presence of microbes in fresh DI water – and suggested a possible source for the “contaminants” in prepared salt solutions. Propensity of microbes for forming biofilm on various surfaces suggested that intermittent flow in just-in-time DI water production provided opportunities for cell attachment and biofilm formation in the system during water stagnation, and subsequent dislodgement and resuspension of cells upon water flow. Thus, regular maintenance and cleaning of the production system should help reduce DI water’s microbial load. Additionally, simple and low-cost culture experiments on agar medium can provide a qualitative and semi-quantitative estimate of microbial diversity and viable cell concentration in DI water, respectively; which, along with regular monitoring of water resistivity or conductivity, comprise a trio of tests useful for detecting possible contamination or, deterioration of DI water’s chemical and microbiological quality.

Microbes in deionized water: Implications for maintenance of laboratory water production system

Microbes, with their diverse metabolic capabilities and great adaptability, occupy almost every conceivable ecological niche on Earth – thus, could they survive in the oligotrophic (i.e., nutrient-poor) deionized (DI) water that we use for our experiments? Observations of white cauliflower-like lumps and black specks in salt solutions after months of storage in plastic bottles prompted the inquisition concerning the origin and nature of the “contaminants.” Hypothesising that the “contaminants” may be microbes from DI water, a series of growth experiments was conducted to detect and profile the microbial diversity in fresh DI water - produced on a just-in-time basis by a filter-cum-ion-exchange system with tap water as feed. While microbes could also be present on the surfaces and headspace of the unsterilized polyethylene bottles, investigating whether microbes are present in freshly produced DI water provides a more stringent performance test of the production system. Inoculation of DI water on R2A agar followed by multi-day aerobic cultivation revealed the presence of a wide variety of microbes (total viable cell concentration of ~103 colony forming units (CFU) per mL) with differing pigmentations, growth rates as well as colony sizes and morphologies. Additionally, greater abundance and diversity of microbes was recovered at 30 oC relative to 25 and 37 oC; most probably due to adaptation of microbes to tropical ambient water temperatures of 25 to 30 oC. Comparative experiments with tap water as inoculum recovered a significantly smaller number and diversity of microbes; thereby, suggesting that monochloramine residual disinfectant in tap water was effective in inhibiting cell viability. In contrast, possible removal of monochloramine by adsorption onto ion-exchange resins – and thus, alleviation of a source of environmental stress - might explain the observed greater diversity and abundance of viable microbes in DI water. Collectively, this study confirmed the presence of microbes in fresh DI water – and suggested a possible source for the “contaminants” in prepared salt solutions. Propensity of microbes for forming biofilm on various surfaces suggested that intermittent flow in just-in-time DI water production provided opportunities for cell attachment and biofilm formation in the system during water stagnation, and subsequent dislodgement and resuspension of cells upon water flow. Thus, regular maintenance and cleaning of the production system should help reduce DI water’s microbial load. Additionally, simple and low-cost culture experiments on agar medium can provide a qualitative and semi-quantitative estimate of microbial diversity and viable cell concentration in DI water, respectively; which, along with regular monitoring of water resistivity or conductivity, comprise a trio of tests useful for detecting possible contamination or, deterioration of DI water’s chemical and microbiological quality.

Active coating to prolong the shelf life of Fior di latte cheese

This study explains how active coating can serve to prolong the shelf life of Fior di latte cheese. The active coating was prepared by dissolving, in two sodium alginic acid solutions (5 and 8% w/v), different concentrations of lysozyme (0·25, 0·50 and 1·00 mg ml−1)+50 mmof Ethylene-Diamine Tetraacetic Acid (EDTA). Samples of Fior di latte cheese packaged in brine and active brine (lysozyme+EDTA, at the above concentrations) were also used as controls. The quality decay of the Fior di latte cheese stored at 10°C was assessed by monitoring the viable cell concentration of the main spoilage microorganism, as well as its sensory quality (i.e., external appearance, consistency, colour and flavour). The concentration of rod-or coccus-shaped Lactic Acid Bacteria (LAB) was also monitored to assess the effect of the proposed packaging strategies on the flora type of Fior di latte cheese. The results show that an increase in the shelf life equal to 104% was recorded for the coated samples, compared with controls packaged in brine without active compounds. This shelf life increase is slightly lower than that recorded with samples packaged in the active brine (151%), as a result of a more pronounced microbial proliferation; however, the coating could be a better packaging solution for the reduced weight of tray.