Unveiling the potential of specific growth rate control in fed-batch fermentation: bridging the gap between product quantity and quality.

During the epoch of sustainable development, leveraging cellular systems for production of diverse chemicals via fermentation has garnered attention. Industrial fermentation, extending beyond strain efficiency and optimal conditions, necessitates a profound understanding of microorganism growth characteristics. Specific growth rate (SGR) is designated as a key variable due to its influence on cellular physiology, product synthesis rates and end-product quality. Despite its significance, the lack of real-time measurements and robust control systems hampers SGR control strategy implementation. The narrative in this contribution delves into the challenges associated with the SGR control and presents perspectives on various control strategies, integration of soft-sensors for real-time measurement and control of SGR. The discussion highlights practical and simple SGR control schemes, suggesting their seamless integration into industrial fermenters. Recommendations provided aim to propose new algorithms accommodating mechanistic and data-driven modelling for enhanced progress in industrial fermentation in the context of sustainable bioprocessing.

Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production.

This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H2S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H2S concentrations. Results suggested that Type II methanotrophs can possess a native H2S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production.

BACKGROUND: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. RESULTS: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. CONCLUSIONS: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation.

BACKGROUND: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products. RESULTS: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane. CONCLUSIONS: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Pilot composite tubular bioreactor for outdoor photo-fermentation hydrogen production: From batch to continuous operation.

A novel 70 L composite tubular photo-bioreactor was constructed, and its photo-fermentation hydrogen production characteristics of batch and continuous modes were investigated with glucose as the substrate in an outdoor environment. In the batch fermentation stage, the hydrogen production rate peaked at 37.6 mL H2/(L·h) accompanied by a high hydrogen yield of 7 mol H2/mol glucose. The daytime light conversion efficiency is 4 %, with 37 % of light energy from the sun. An optimal hydraulic retention time of 5 d was identified during continuous photo-fermentation. Under this condition, the stability of the cell concentration is maintained and more electrons can be driven to the hydrogen generation pathway while attaining a hydrogen production rate of 20.7 ± 0.9 mL H2/(L·h). The changes of biomass, volatile fatty acids concentration and ion concentration during fermentation were analyzed. Continuous hydrogen production by composite tubular photo-bioreactor offers new ideas for the large-scale deployment of photobiological hydrogen production.

Enhanced high ß-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals.

This study assessed Rhodotorula paludigena CM33's growth and ß-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and ß-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a ß-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Qx) of 0.91 g/L.h and remarkable ß-carotene production rates (Qp) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale ß-carotene production for aquatic animal nutrition.

Continuous catalytic production of 1,3-dihydroxyacetone: Sustainable approach combining perfusion cultures and immobilized cells.

Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research has revealed that in the biocatalytic synthesis of DHA from glycerol, when the DHA concentration exceeded 50 g·L-1, it significantly inhibited microbial growth and metabolism, posing a challenge in maintaining prolonged and efficient catalytic production of DHA. In this study, a new integrated continuous production and synchronous separation (ICSS) system was constructed using hollow fiber columns and perfusion culture technology. Additionally, a cell reactivation technique was implemented to extend the biocatalytic ability of cells. Compared with fed-batch fermentation, the ICSS system operated for 360 h, yielding a total DHA of 1237.8 ± 15.8 g. The glycerol conversion rate reached 97.7 %, with a productivity of 3.44 g·L-1·h-1, representing 485.0 % increase in DHA production. ICSS system exhibited strong operational characteristics and excellent performance, indicating significant potential for applications in industrial bioprocesses.

Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Platform development for high-throughput optimization of perfusion processes-Part II: Variation of perfusion rate strategies in microwell plates.

The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell-specific perfusion rate (CSPR). To obtain a cost-productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor-intensive and time-consuming experiments, as investigations are performed in lab-scale perfusion bioreactors. Small-scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi-perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi-perfusion. The CSPR-based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d-1. The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 106 cells mL-1 were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR-based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations.

Efficient conversion of cane molasses into Tremella fuciformis polysaccharides with enhanced bioactivity through repeated batch culture.

Tremella fuciformis polysaccharide (TFPS) is a natural mushroom mucopolysaccharide widely used in health foods, medical care, cosmetic and surgical materials. In this study, we developed an efficient strategy for the repeated batch production of highly bioactive TFPS from the agro-industrial residue cane molasses. Cane molasses contained 39.92 % sucrose (w/w), 6.36 % fructose and 3.53 % glucose, all of which could be utilized by T. fuciformis spores, whereas, the TFPS production efficiency only reached 0.74 g/L/d. Corn cobs proved to be the best immobilized carrier that could tightly absorb spores and significantly shorten the fermentation lag period. The average yield of TFPS in eight repeated batch culture was 5.52 g/L with a production efficiency of 2.04 g/L/d. The average fermentation cycle after optimization was reduced by 61.61 % compared with the initial conditions. Compared to glucose as a carbon source, cane molasses significantly increased the proportion of low-molecular-weight TFPS (TFPS-2) in total polysaccharides from 3.54 % to 17.25 % (w/w). Moreover, TFPS-2 exhibited potent antioxidant capacity against four free radicals (O2-, ABTS+, OH, and DPPH). In conclusion, this study lays the foundation for the efficient conversion of cane molasses and production of TFPS with high bioactivity.

Robust platform for inline Raman monitoring and control of perfusion cell culture.

Perfusion cell culture has been gaining increasing popularity for biologics manufacturing due to benefits such as smaller footprint, increased productivity, consistent product quality and manufacturing flexibility, cost savings, and so forth. Process Analytics Technologies tools are highly desirable for effective monitoring and control of long-running perfusion processes. Raman has been widely investigated for monitoring and control of traditional fed batch cell culture process. However, implementation of Raman for perfusion cell culture has been very limited mainly due to challenges with high-cell density and long running times during perfusion which cause extremely high fluorescence interference to Raman spectra and consequently it is exceedingly difficult to develop robust chemometrics models. In this work, a platform based on Raman measurement of permeate has been proposed for effective analysis of perfusion process. It has been demonstrated that this platform can effectively circumvent the fluorescence interference issue while providing rich and timely information about perfusion dynamics to enable efficient process monitoring and robust bioreactor feed control. With the highly consistent spectral data from cell-free sample matrix, development of chemometrics models can be greatly facilitated. Based on this platform, Raman models have been developed for good measurement of several analytes including glucose, lactate, glutamine, glutamate, and permeate titer. Performance of Raman models developed this way has been systematically evaluated and the models have shown good robustness against changes in perfusion scale and variations in permeate flowrate; thus models developed from small lab scale can be directly transferred for implementation in much larger scale of perfusion. With demonstrated robustness, this platform provides a reliable approach for automated glucose feed control in perfusion bioreactors. Glucose model developed from small lab scale has been successfully implemented for automated continuous glucose feed control of perfusion cell culture at much larger scale.

Two at once: simultaneous increased production of astaxanthin and mycosporines in a single batch culture using a Phaffia rhodozyma mutant strain.

Phaffia rhodozyma is a basidiomycetous yeast characterized by its production of the carotenoid pigment astaxanthin, which holds high commercial value for its significance in aquaculture, cosmetics and as nutraceutics, and the UV-B-absorbing compound mycosporine-glutaminol-glucoside (MGG), which is of great biotechnological relevance for its incorporation into natural sunscreens. However, the industrial exploitation has been limited to the production of astaxanthin in small quantities. On the other hand, the accumulation of MGG in P. rhodozyma was recently reported and could add value to the simultaneous production of both metabolites. In this work, we obtain a mutant strain that overproduces both compounds, furthermore we determined how the accumulation of each is affected by the carbon-to-nitrogen ratio and six biotic and abiotic factors. The mutant obtained produces 159% more astaxanthin (470.1 µg g-1) and 220% more MGG (57.9 mg g-1) than the parental strain (295.8 µg g-1 and 26.2 mg g-1 respectively). Furthermore, we establish that the carotenoids accumulate during the exponential growth phase while MGG accumulates during the stationary phase. The carbon-to-nitrogen ratio affects each metabolite differently, high ratios favoring carotenoid accumulation while low ratios favoring MGG accumulation. Finally, the accumulation of both metabolites is stimulated only by photosynthetically active radiation and low concentrations of hydrogen peroxide. The mutant strain obtained is the first hyper-productive mutant capable of accumulating high concentrations of MGG and astaxanthin described to date. The characterization of how both compounds accumulate during growth and the factors that stimulate their accumulation, are the first steps toward the future commercial exploitation of strains for the simultaneous production of two biotechnologically important metabolites.

Dynamic flux balance analysis of high cell density fed-batch culture of Escherichia coli BL21 (DE3) with mass spectrometry-based spent media analysis.

Dynamic flux balance analysis (FBA) allows estimation of intracellular reaction rates using organism-specific genome-scale metabolic models (GSMM) and by assuming instantaneous pseudo-steady states for processes that are inherently dynamic. This technique is well-suited for industrial bioprocesses employing complex media characterized by a hierarchy of substrate uptake and product secretion. However, knowledge of exchange rates of many components of the media would be required to obtain meaningful results. Here, we performed spent media analysis using mass spectrometry coupled with liquid and gas chromatography for a fed-batch, high-cell density cultivation of Escherichia coli BL21(DE3) expressing a recombinant protein. Time course measurements thus obtained for 246 metabolites were converted to instantaneous exchange rates. These were then used as constraints for dynamic FBA using a previously reported GSMM, thus providing insights into how the flux map evolves through the process. Changes in tri-carboxylic acid cycle fluxes correlated with the increased demand for energy during recombinant protein production. The results show how amino acids act as hubs for the synthesis of other cellular metabolites. Our results provide a deeper understanding of an industrial bioprocess and will have implications in further optimizing the process.

Mimicking CHO large-scale effects in the single multicompartment bioreactor: A new approach to access scale-up behavior.

During the scale-up of biopharmaceutical production processes, insufficiently predictable performance losses may occur alongside gradients and heterogeneities. To overcome such performance losses, tools are required to explain, predict, and ultimately prohibit inconsistencies between laboratory and commercial scale. In this work, we performed CHO fed-batch cultivations in the single multicompartment bioreactor (SMCB), a new scale-down reactor system that offers new access to study large-scale heterogeneities in mammalian cell cultures. At volumetric power inputs of 20.4-1.5 W m-3, large-scale characteristics like long mixing times and dissolved oxygen (DO) heterogeneities were mimicked in the SMCB. Compared to a reference bioreactor (REFB) set-up, the conditions in the SMCB provoked an increase in lactate accumulation of up to 87%, an increased glucose uptake, and reduced viable cell concentrations in the stationary phase. All are characteristic for large-scale performance. The unique possibility to distinguish between the effects of changing power inputs and observed heterogeneities provided new insights into the potential reasons for altered product quality attributes. Apparently, the degree of galactosylation in the evaluated glycan patterns changed primarily due to the different power inputs rather than the provoked heterogeneities. The SMCB system could serve as a potent tool to provide new insights into scale-up behavior and to predict cell line-specific drawbacks at an early stage of process development.

Exploring metabolic effects of dipeptide feed media on CHO cell cultures by in silico model-guided flux analysis.

There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer. KEY POINTS: • Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance • Systematically analyzed effects of dipeptide media by model-guided approach • Uncovered synergistic metabolic utilization of amino acids with dipeptide.

Development of an in-line Raman analytical method for commercial-scale CHO cell culture process monitoring: Influence of measurement channels and batch number on model performance.

The mammalian cell culture process is a key step in commercial therapeutic protein production and needs to be monitored and controlled due to its complexity. Raman spectroscopy has been reported for cell culture process monitoring by analysis of many important parameters. However, studies on in-line Raman monitoring of the cell culture process were mainly conducted on small or pilot scale. Developing in-line Raman analytical methods for commercial-scale cell culture process monitoring is more challenging. In this study, an in-line Raman analytical method was developed for monitoring glucose, lactate, and viable cell density (VCD) in the Chinese hamster ovary (CHO) cell culture process during commercial production of biosimilar adalimumab (1500 L). The influence of different Raman measurement channels was considered to determine whether to merge data from different channels for model development. Raman calibration models were developed and optimized, with minimum root mean square error of prediction of 0.22 g L-1 for glucose in the range of 1.66-3.53 g L-1 , 0.08 g L-1 for lactate in the range of 0.15-1.19 g L-1 , 0.31 E6 cells mL-1 for VCD in the range of 0.96-5.68 E6 cells mL-1 on test sets. The developed analytical method can be used for cell culture process monitoring during manufacturing and meets the analytical purpose of this study. Further, the influence of the number of batches used for model calibration on model performance was also studied to determine how many batches are needed basically for method development. The proposed Raman analytical method development strategy and considerations will be useful for monitoring of similar bioprocesses.

Intensified perfusion culture (IPC) reduced recombinant protein fragmentation.

Mammalian cells remain the mainstay of biological production host. In industry, cultivating and harvest strategies are sorted in batch mode (e.g., batch, fed-batch, concentrated fed-batch and intensified fed-batch) and continuous mode (e.g., perfusion). To retrieve greater productivity and better product quality, especially for the sensitive products prone to fragmentation, culture modes with various modifications are innovated (e.g., intensified perfusion culture [IPC]). In our study, we demonstrated that the fragmentation of Fc-fusion product (Molecule A) is time-dependent in traditional fed-batch (TFB) culture. The fragmentation proportion increased from 3.8% to 12.4% for Clone A, 0.8% to 1.7% for Clone B and 0.9% to 2.0% for Clone C from Day 10 to Day 14. By applying a novel bioprocess, IPC, which allows continuous feeding of the fresh medium and constant removal of the spent medium without bleeding cells to maintain a defined constant viable cell density, the fragmentation was reduced to 0.3% while the productivity was increased from 2.96 g/L to 15.51 g/L for Clone A. To validate whether the fragmentation reduction is product-sensitive, plasmids carrying the DNA sequences of two other Fc-fusion molecules (Molecule B and Molecule C) were transfected into the host. The results showed consistent fragmentation reducing effect by using IPC. Furthermore, the cultivation scale was expanded to 50 L and 1000 L. A minimum fragmentation level below 0.1% was observed for Molecule C. Our study revealed the capability of IPC in reducing Fc-fusion protein fragmentation and the reproducibility when scaling up while maintaining high productivity.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus-1.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 108 TCID50 mL-1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 106 cells mL-1 , and was further improved to 1.1 × 109 TCID50 mL-1 in perfusion culture infected at 4.6 × 106 cells mL-1 . These titers are similar to or better than the previously reported best titer of 8.6 × 107 TCID50 mL-1 and 8.1 × 108 TCID50 mL-1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes.