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.

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.

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.

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.

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.

Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Effect of adenosine and cordycepin on recombinant antibody production yields in two different Chinease hamster ovary cell lines.

In this study, we investigated the effect of adenosine and its derivative cordycepin on the production yield of a recombinant human monoclonal antibody (adalimumab) in two commonly used Chinese Hamster ovary (CHO) cell lines that have different gene amplification systems, namely CHO-DHFR- and GS-CHO knockout (GS-KO CHO) cells and that were grown in batch culture, with and without glucose feeding. The results showed that adenosine suppressed the cell growth rate and increased the fraction of cells in S phase of the cell cycle for both CHO cell lines. Different concentrations and exposure times of adenosine feeding were tested. The optimal yield of adalimumab production was achieved with the addition of 1 mM adenosine on day 2 after start of the batch culture. Adenosine could significantly improve antibody titers and productivity in both CHO cell lines in cultures without glucose feeding. However, upon glucose feeding, adenosine did not improve antibody titers in CHO-DHFR- cells but extended culture duration and significantly increased antibody titers in GS-KO CHO cells. Therefore, adenosine supplementation might be useful for antibody production in GS-KO CHO cells in medium- to large-scale batches. In case of cordycepin, a derivative of adenosine, CHO-DHFR- cells required higher concentration of cordycepin than GS-KO CHO cells around 10 times to display the changes in cell growth and cell cycle. Moreover, cordycepin could significantly increase antibody titers only in CHO-DHFR- cell cultures without glucose feeding.

Developing an ultra-intensified fed-batch cell culture process with greatly improved performance and productivity.

Intensified fed-batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed-batch cultivation. However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult-to-sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent-perfusion fed-batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi-continuous processes such as hybrid perfusion fed-batch with only early-stage perfusion, IPFB applies limited times of intermittent perfusion in the mid-to-late stage of production and still inherits bolus feedings on nonperfusion days as in a fed-batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra-intensified IPFB (UI-IPFB) was designed with a markedly elevated seeding density of 20-80 × 106 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI-IPFB, up to ~6 folds of traditional fed-batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad-based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities.

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.

The stressostat: A novel approach in adaptive laboratory evolution to improve end-product resistance.

End-product inhibition in pH-controlled batch cultures, is the major limiting factor for bacterial biomass formation in the starter culture industry as well as in many other biotechnological processes. Adaptive laboratory evolution (ALE) has emerged over the past decades as a powerful tool for phenotype optimization, but none of the existing ALE methods could select for improved end-product resistance. Therefore, we developed the stressostat (STress Resistance Evolution in Substrate Surplus) as a novel continuous ALE method. Stressostat cultivation applies end-product concentrations as constant evolutionary pressure on microorganisms in the presence of substrate surplus. In this study, we improved the lactate resistance of Lactococcus lactis FM03P in 35 days of stressostat cultivations. The lactate concentrations increased over time from 530 to 675 mM, indicating the successful selection for variants with improved lactate resistance. Thirty-four variants were isolated and grouped into four clusters based on their growth rates at high lactate concentrations. In the high-throughput screening without pH control, most isolated variants could grow at high lactate concentrations (870-928 mM), while the wild type was completely inhibited. The variants grew slower than wild type at low lactate media indicating possible evolutionary trade-off. However, in pH-controlled batch cultivations, most variants produced more biomass than the wild type. In conclusion, stressostat cultivation is a valuable method to obtain L. lactis variants with improved end-product resistance and further characterization is needed to elucidate underlying resistance mechanisms and potential industrial applications.

A holistic approach for process intensification of nicotinamide mononucleotide production via high cell density cultivation under exponential feeding strategy.

Nicotinamide mononucleotide (NMN) subsists in all living organisms and has drawn tremendous attention as a nutraceutical and pharmaceutical product for several diseases such as Alzheimer's, cancer, aging, and vascular dysfunction. Here, NMN was produced intracellularly in a high cell density bioreactor using an engineered Escherichiacoli strain via exponential feeding of co-substrates. Fed-batch culture via exponential feeding of co-substrate (glucose) and continuous feeding of substrate (nicotinamide) were performed using different cumulative nicotinamide concentrations. The highest concentration of 19.3 g/L NMN with a dry cell weight of 117 g/L was acquired from a cumulative nicotinamide concentration of 7.2 g/L with a conversion of 98 % from nicotinamide in 28 h. Further, liquid chromatography-mass spectrometry analysis validated the NMN production. This approach will be beneficial in achieving simultaneously low cost and ensuring high quality and quantity of NMN production.

Risk assessment for biopharmaceutical single-use manufacturing: A case study of upstream continuous processing.

In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.

Batch culture analysis to identify potent organic acids for suppressing ruminal methane production.

We performed an in vitro rumen batch culture study to screen 11 commercially available organic acids for methane-suppressing ability and analyzed the rumen microbiota to determine the mode of action of the acids that showed potent methane-suppressing activity. Nine of the 11 acids showed methane-suppressing activity. Maleic anhydride, itaconate, citrate, and fumarate, which showed the highest activity, were further examined. These four acids showed methane-suppressing activity irrespective of the hay-to-concentrate ratios of the substrate. Maleic anhydride and itaconate decreased total gas and short-chain fatty acid production. Maleic anhydride and fumarate increased propionate production, while itaconate increased butyrate production. Maleic anhydride, itaconate, and citrate increased lactate production. Fumarate increased the abundance of bacteria involved in propionate production. Maleic anhydride, itaconate, and citrate increased the abundance of bacteria involved in lactate production. Thus, the results indicate that maleic anhydride, itaconate, and citrate may decrease methane in part by stimulating the acrylate pathway.

The harmful cyanobacterium Microcystis aeruginosa PCC7806 is more resistant to hydrogen peroxide at elevated CO2.

Rising atmospheric CO2 can intensify harmful cyanobacterial blooms in eutrophic lakes. Worldwide, these blooms are an increasing environmental concern. Low concentrations of hydrogen peroxide (H2O2) have been proposed as a short-term but eco-friendly approach to selectively mitigate cyanobacterial blooms. However, sensitivity of cyanobacteria to H2O2 can vary depending on the available resources. To find out how cyanobacteria respond to H2O2 under elevated CO2, Microcystis aeruginosa PCC 7806 was cultured in chemostats with nutrient-replete medium under C-limiting and C-replete conditions (150 ppm and 1500 ppm CO2, respectively). Microcystis chemostats exposed to high CO2 showed higher cell densities, biovolumes, and microcystin contents, but a lower photosynthetic efficiency and pH compared to the cultures grown under low CO2. Subsamples of the chemostats were treated with different concentrations of H2O2 (0-10 mg·L-1 H2O2) in batch cultures under two different light intensities (15 and 100 µmol photons m-2·s-1) and the response in photosynthetic vitality was monitored during 24 h. Results showed that Microcystis was more resistant to H2O2 at elevated CO2 than under carbon-limited conditions. Both low and high CO2-adapted cells were more sensitive to H2O2 at high light than at low light. Microcystins (MCs) leaked out of the cells of cultures exposed to 2-10 mg·L-1 H2O2, while the sum of intra- and extracellular MCs decreased. Although both H2O2 and CO2 concentrations in lakes vary in response to many factors, these results imply that it may become more difficult to suppress cyanobacterial blooms in eutrophic lakes when atmospheric CO2 concentrations continue to rise.

Transcriptomics analysis and fed-batch regulation of high astaxanthin-producing Phaffia rhodozyma/Xanthophyllomyces dendrorhous obtained through adaptive laboratory evolution.

Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma. ONE-SENTENCE SUMMARY: A high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.

[Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells].

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

Transcriptomics and cell painting analysis reveals molecular and morphological features associated with fed-batch production performance in CHO recombinant clones.

Stable, highly productive mammalian cells are critical for manufacturing affordable and effective biological medicines. Establishing a rational design of optimal biotherapeutic expression systems requires understanding how cells support the high demand for efficient biologics production. To that end, we performed transcriptomics and high-throughput imaging studies to identify putative genes and morphological features that underpin differences in antibody productivity among clones from a Chinese hamster ovary cell line. During log phase growth, we found that the expression of genes involved in biological processes related to cellular morphology varied significantly between clones with high specific productivity (qP > 35 pg/cell/day) and low specific productivity (qP < 20 pg/cell/day). At Day 10 of a fed-batch production run, near peak viable cell density, differences in gene expression related to metabolism, epigenetic regulation, and proliferation became prominent. Furthermore, we identified a subset of genes whose expression predicted overall productivity, including glutathione synthetase (Gss) and lactate dehydrogenase A (LDHA). Finally, we demonstrated the feasibility of cell painting coupled with high-throughput imaging to assess the morphological properties of intracellular organelles in relation to growth and productivity in fed-batch production. Our efforts lay the groundwork for systematic elucidation of clone performance using a multiomics approach that can guide future process design strategies.