Cell Culture Production Research Articles

Effects of Succinic Acid Supplementation on Stable Cell Line Growth, Aggregation, and IgG and IgA Production.

Immunoglobulin (Ig) G is the most commonly used therapeutic antibodies. Recently, the interest in IgA antibodies to treat respiratory infectious diseases has been increasing. The reason for the inefficient use of IgA is recombinant antibody aggregation in cell culture, affecting the longevity and productivity of cell lines. Lactate is an important metabolite that affects the cultivation of stable cell lines producing monoclonal antibodies. In the present study, we investigated whether different combinations of succinic acid and micro-additives affect lactate production, which correlates with productivity. The effect of succinic acid substitution on productivity of cells producing IgG/IgA was analyzed using the static culture method in a six-well plate. Lactate was measured in supernatant of cell culture indirectly by using the activity of Lactate Dehydrogenase (LDH).A low lactate level was observed in cultivation medium supplemented with succinic acid or asparagine combined with some inorganic salts. The results also demonstrated the effect of component supplementation on homogeneity, longevity, and productivity of cell culture. Supplementation of succinic acid eliminated cell aggregation and improved homogeneity of stable cell lines producing IgG and, especially, IgA. Overall, succinic acid supplementation to the culture medium has potential biotechnological applications in the production IgG and IgA.

Modeling of paclitaxel biosynthesis elicitation in Corylus avellana cell culture using adaptive neuro-fuzzy inference system-genetic algorithm (ANFIS-GA) and multiple regression methods.

Paclitaxel as a microtubule-stabilizing agent is widely used for the treatment of a vast range of cancers. Corylus avellana cell suspension culture (CSC) is a promising strategy for paclitaxel production. Elicitation of paclitaxel biosynthesis pathway is a key approach for improving its production in cell culture. However, optimization of this process is time-consuming and costly. Modeling of paclitaxel elicitation process can be helpful to predict the optimal condition for its high production in cell culture. The objective of this study was modeling and forecasting paclitaxel biosynthesis in C. avellana cell culture responding cell extract (CE), culture filtrate (CF) and cell wall (CW) derived from endophytic fungus, either individually or combined treatment with methyl-β-cyclodextrin (MBCD), based on four input variables including concentration levels of fungal elicitors and MBCD, elicitor adding day and CSC harvesting time, using adaptive neuro-fuzzy inference system (ANFIS) and multiple regression methods. The results displayed a higher accuracy of ANFIS models (0.94–0.97) as compared to regression models (0.16–0.54). The great accordance between the predicted and observed values of paclitaxel biosynthesis for both training and testing subsets support excellent performance of developed ANFIS models. Optimization process of developed ANFIS models with genetic algorithm (GA) showed that optimal MBCD (47.65 mM) and CW (2.77% (v/v)) concentration levels, elicitor adding day (16) and CSC harvesting time (139 h and 41 min after elicitation) can lead to highest paclitaxel biosynthesis (427.92 μg l-1). The validation experiment showed that ANFIS-GA method can be a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis, as a case study.

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3−/−) the spleen’s histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3−/− mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3−/− and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3−/− mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3−/− mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1β, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3−/− mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Synthesis, antioxidant properties and neuroprotection of \u03b1-phenyl-tert-butylnitrone derived HomoBisNitrones in in vitro and in vivo ischemia models

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs1–9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs1–9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1′Z)-1,1′-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50 = 1.24 ± 0.39 μM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity = 95.8 ± 3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.

Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins.

Immobilized metal affinity chromatography (IMAC) is a technique primarily used in research and development laboratories to purify proteins containing engineered histidine tags. Although this type of chromatography is commonly used, it can be problematic as differing combinations of resins and metal chelators can result in highly variable chromatographic performance and product quality results. To generate a robust IMAC purification process, the binding differences of resin and metal chelator combinations were studied by generating breakthrough curves with a poly-histidine tagged bispecific protein. The optimal binding combination was statistically analyzed to determine the impact of chromatographic parameters on the operation. Additionally, equilibrium uptake isotherms were created to further elucidate the impact of chromatographic parameters on the binding of protein. It was found that for protein expressed in CHO cells, Millipore Sigma's Fractogel EMD Chelate (M) charged with Zn2+ and GE's pre-charged Ni Sepharose Excel displayed the highest binding capacities. When the protein was expressed in HEK-293, GE's IMAC Sepharose 6 Fast Flow charged with either Co2+ or Zn2+ bound the greatest amount of protein. The study further identified the metal binding capacity of the resin lot, the protein capacity to which the resin is loaded, and the ratio of poly-histidine tag residues on the protein all impacted the chromatographic performance and product quality. These findings enabled the development of a robust and scalable process. The CHO expressed cell culture product was directly loaded at a high capacity onto variable metal binding affinity Fractogel EMD Chelate (M). A 250 mM imidazole elution condition ensured the product contained monomeric 4 and 6-histidine tagged bispecific proteins. The optimized IMAC process conditions determined in this study can be applied to a wide variety of poly-histidine tagged proteins in research and development laboratories as various poly-histidine tagged proteins of differing molecular weights and formats expressed in either HEK-293 or CHO cells were successfully purified.

Forskolin Increases cAMP Levels and Enhances Recombinant Antibody Production in CHO Cell Cultures

AbstractCyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger essential to many biological processes. Forskolin, a labdane diterpene, is widely used to increase cAMP levels in various cell types. When 5 µm of forskolin is added to recombinant Chinese hamster ovary (rCHO) cell cultures producing monoclonal antibody (mAb) (GSR cell line), it decreases specific growth rate (μ), but increases culture longevity and specific mAb productivity (qmAb). The beneficial effect of forskolin on culture longevity and qmAb outweighs its detrimental effect on μ, resulting in 1.5‐fold increase in the maximum mAb concentration (MMC) without an adverse effect on mAb quality attributes such as aggregation, charge variation, and galactosylation. Forskolin induces cell cycle arrest at the G0/G1 phase via the LKB1/AMPK pathway and inhibits apoptosis via the CREB/Bcl‐2 pathway. The beneficial effect of forskolin on mAb production is also demonstrated with another rCHO cell line (ABTAA). Addition of 5 µm forskolin to ABTAA cell cultures also results in 1.5‐fold increase in the MMC. Taken together, the results obtained demonstrate that forskolin is an efficient chemical reagent to increase mAb production in rCHO cell cultures.

Quality-targeting dynamic optimization of monoclonal antibody production

Compliance with Quality by Design (QbD) constitutes a major challenge in biopharmaceuticals. Monoclonal antibodies (mAbs) represent a significant biopharmaceutical product class, typically produced in mammalian cell cultures. A key quality attribute for mAb production is glycosylation. We examine how process intensification affects glycosylation via dynamic optimization using different problem formulations. We maximize process performance with simultaneous control of product quality. For these, we utilize a mechanistic dynamic model for mAb production in mammalian cell cultures including glycosylation presented by Ehsani et al. in Computer Aided Chemical Engineering (2017). To achieve target glycan distribution in the final product, we incorporate constraints for the acceptable glycosylation ranges into the dynamic optimization problem. As a result, we derive optimal supplementation profiles of nutrients and/or nucleotide sugars. This work successfully illustrates an example of how model-based dynamic optimization can be employed for implementation of the QbD approach in biopharmaceutics.

The life cycle of cyclotides: biosynthesis and turnover in plant cells

Key messageTurnover rates have implications for understanding cyclotide biology and improving plant cell culture-based production systems.Cyclotides are a family of polypeptides recognized for a broad spectrum of bioactivities. The cyclic, cystine knot structural motif imparts these peptides with resistance to temperature, chemicals and proteolysis. Cyclotides are found widely distributed across the Violaceae and in five other plant families, where their presumed biological role is host defense. Violets produce mixtures of different cyclotides that vary depending on the organ, tissue or influence of environmental factors. In the present study, we investigated the biosynthesis and turnover of cyclotides in plant cells. Viola uliginosa suspension cultures were grown in media where all nitrogen containing salts were replaced with their 15N counterparts. This approach combined with LC–MS analysis allowed to separately observe the production of 15N-labelled peptides and decomposition of 14N cyclotides present in the cells when switching the media. Additionally, we investigated changes in cyclotide content in V. odorata germinating seeds. In the suspension cultures, the degradation rates varied for individual cyclotides and the highest was noted for cyO13. Rapid increase in production of 15N peptides was observed until day 19 and subsequently, a plateau of production, indicating an equilibrium between biosynthesis and turnover. The developing seedling appeared to consume cyclotides present in the seed endosperm. We show that degradation processes shape the cyclotide pattern present in different tissues and environments. The results indicate that individual cyclotides play different roles—some in defense and others as storage proteins. The turnover of cyclotides should be accounted to improve cell culture production systems.

Antihypoxic Action of Panax Japonicus, Tribulus Terrestris and Dioscorea Deltoidea Cell Cultures: In Silico and Animal Studies.

Chemical diversity of secondary metabolites provides a considerable variety of pharmacological actions with a significant extension due to their combinations in plant extracts. Production of plant-derived medicinal products in cell cultures has advantages because of the efficient use of different biotic and abiotic elicitors and better control of the developmental processes. Using PASS software, we predicted biological activity spectra for phytoconstituents identified in cell cultures of Panax japonicus (12 molecules), Tribulus terrestris (4 molecules), and Dioscorea deltoidea (3 molecules). Mechanisms of action associated with the antihypoxic effect were predicted for the majority of molecules. PharmaExpert software allowed analyzing possible synergistic or additive effects of the combinations of phytoconstituents associated with the antihypoxic action. Experimental studies of the antihypoxic effect of the plants' extracts in water and ethanol have been performed in 3 animal models: Acute asphyctic hypoxia (AAH), Acute haemic hypoxia (AHeH), and Acute histotoxic hypoxia (AHtH). Effects of Panax japonicus and Tribulus terrestris preparations exceeded the activity of the reference drug Mexidol in the AHtH model. In the AHeH model, all preparations demonstrated moderate activity; the most potent has been observed for Dioscorea deltoidea. Thus, we found that experimental studies in animal models have confirmed the in silico prediction.

Induced extracellular production of stilbenes in grapevine cell culture medium by elicitation with methyl jasmonate and stevioside

We report the high production of stilbenes, including resveratrol and viniferin, in grapevine (Vitis labruscana L.) cell cultures through elicitation with methyl jasmonate (MeJA) and stevioside (STE). Methyl-β-cyclodextrin (MeβCD) is widely used as a solubilizer for resveratrol production. For the first time, we used STE as a solubilizer for stilbene production in plant cell cultures. MeJA was most effective elicitor in activating VvSTS expression and stimulating stilbene biosynthesis in grapevine cell cultures. The maximum concentration of δ-viniferin (892.2 mg/L) production with a small amount of trans-resveratrol (12.2 mg/L) was observed in the culture medium by co-treatment of cells with MeJA and STE, whereas the highest level of trans-resveratrol (371.9 mg/L) with a slight amount of δ-viniferin (11.5 mg/L) was accumulated in the culture medium of cells treated with MeJA and MeβCD. However, neither trans-resveratrol nor δ-viniferin were significantly elevated within the cells by the applications. Notably, predominant production of δ-viniferin and trans-resveratrol was observed in shake and static flask culture medium, respectively, by co-treatment of MeJA and STE. Furthermore, stilbene compounds of resveratrol, ε-viniferin, and δ-viniferin were mainly produced in a 3-L bioreactor culture following elicitation of cells with MeJA and STE. These results provide new strategies for conditional, high-level production of resveratrol and viniferin in cell cultures by utilizing the solubilizing properties of STE or MeβCD.

Genetic and epigenetic dynamics affecting anthocyanin biosynthesis in potato cell culture

Anthocyanins are antioxidant pigments widely used in drugs and food preparations. Flesh-coloured tubers of the cultivated potato Solanum tuberosum are important sources of different anthocyanins. Due to the high degree of decoration achieved by acylation, anthocyanins from potato are very stable and suitable for the food processing industry. The use of cell culture allows to extract anthocyanins on-demand, avoiding seasonality and consequences associated with land-based-tuber production. However, a well-known limit of cell culture is the metabolic instability and loss of anthocyanin production during successive subcultures. To get a general picture of mechanisms responsible for this instability, we explored both genetic and epigenetic regulation that may affect anthocyanin production in cell culture. We selected two clonally related populations of anthocyanin-producing (purple) and non-producing (white) potato cells. Through targeted molecular investigations, we identified and functionally characterized an R3-MYB, here named StMYBATV. This transcription factor can interact with bHLHs belonging to the MBW (R2R3-MYB, bHLH and WD40) anthocyanin activator complex and, potentially, may interfere with its formation. Genome methylation analysis revealed that, for several genomic loci, anthocyanin-producing cells were more methylated than clonally related white cells. In particular, we localized some methylation events in ribosomal protein-coding genes. Overall, our study explores novel molecular aspects associated with loss of anthocyanins in cell culture systems.

Antioxidant Activity and Flavonoid Content of Matricaria Chamomilla Extracts from Different Populations of Iran

Matricaria chamomilla is a valuable medicinal plant belonging to the Asteraceae family. Its medicinal and pharmaceutical impacts are correlated to major flavonoid compounds like apigenin. In this investigation, methanolic extracts of M. chamomilla inflorescence gathered from six natural populations were evaluated for their phytochemical content and antioxidant activity. The content of total flavonoid and phenol modified from 3.72 to 7.94 mg g-1 DW and 1.37 to 3.51 mg g-1 DW, respectively. Flavonoid compositions revealed significant differences among six populations, and the highest apigenin (1.27 % (w/w)) and apigenin-7-glucoside (0.86 %(w/w)) contents were recognized in MD populations, respectively. Both PCA and Pearson's correlation analyses revealed total phenol, flavonoid, apigenin and apigenin-7-glucoside were negatively correlated with the IC50 of DPPH activity and EC50 of reducing power. Altitude and precipitation indicated the positive and negative effects on phytochemical contents, respectively. These results can provide a theoretical basis for getting the targeted antioxidant phytochemicals of M. chamomilla for pharmaceutical and food industries, and also give a science for selection of the best population for cell culture and secondary metabolite production in future.

Technical note: improving the efficiency of generating bovine extraembryonic endoderm cells.

The formation of extraembryonic endoderm (XEN) occurs early in embryonic development. The cell types that develop from the XEN remain poorly studied in ruminant species because of the lack of suitable cell culture model systems. The goal of this work was to establish a protocol for producing XEN cell cultures from bovine blastocysts. Previous work identified fibroblast growth factor 2 (FGF2) as a facilitator of bovine XEN development. Further refinements in culture conditions studied here included exposure to 20% fetal bovine serum and FGF2 replenishment. These modifications yielded an endoderm outgrowth formation incidence of 81.6% ± 5.5% compared with 33.3% ± 5.5% in bovine serum albumin (BSA)-supplemented controls. These cells resembled XEN when examined morphologically and contained XEN transcripts (GATA binding protein 4 [GATA4] and GATA binding protein 6 [GATA6]) as well as transcripts present in visceral (BCL2 interacting protein 1 [BNIP1] and vascular endothelial growth factor A [VEGFA]) and parietal (C-X-C motif chemokine receptor 4 [CXCR4], thrombomodulin [THBD], and hematopoietically expressed homeobox [HHEX]) XEN. Two XEN cell lines were maintained for prolonged culture. Both lines continued to proliferate for approximately 6 wk before becoming senescent. These cultures maintained an XEN-like state and continued to express GATA4 and GATA6 until senescence. An increase in the abundance of visceral and parietal XEN transcripts was observed with continued culture, suggesting that these cells either undergo spontaneous differentiation or retain the ability to form various XEN cell types. Stocks of cultured cells exposed to a freeze-thaw procedure possessed similar phenotypic and genotypic behaviors as nonfrozen cells. To conclude, a procedure for efficient production of primary bovine XEN cell cultures was developed. This new protocol may assist researchers in exploring this overlooked cell type for its roles in nutrient supply during embryogenesis.

Comparative study for the production of monoclonal antibodies in single‐use vs stainless steel bioreactors based on product quality and stress factor

AbstractSingle‐use bioreactors (SUB) and stainless steel bioreactors (SSB) differ in their physical properties. This can have an effect on the performed processes. To be able to guarantee flexible planning in a facility, any kind of processes in SUB and SSB must be sufficiently similar. To date, assessments of SUB and SSB have been exclusively specific for one single product. This study shows a general nonproduct‐specific comparison of the production of different types of monoclonal antibodies in different scales of SUBs and SSBs. The study is set up with 72 clinical manufacturing fermentation runs in total by comparing the cell culture performance and quality attributes of these two bioreactor systems. The outcome is that SUBs and SSBs can produce monoclonal antibodies with comparable cell culture performance and product quality. This general comparability paves the way for SUBs to produce new and existing products for (clinical) manufacturing.

Use of cyclodextrins to improve the production of plant bioactive compounds

Cyclodextrins are cyclic oligosaccharides consisting of d-glucopyranose units bound via α-(1,4)-glycosidic linkages. They are obtained from starch enzymatic degradation by the action of cyclodextrinases or glycosyltransferases. Among the modified-cyclodextrins, 2-hydroxypropylated and methylated β-cyclodextrins are produced on industrial scale. Particularly, methylated β-cyclodextrins are more suitable than native β-cyclodextrins to form stable inclusion complexes with organic molecules of low molecular weight, making these complexes more soluble in aqueous solutions. Cyclodextrins have often been considered useful carriers of antitumor- and immuno-regulatory drugs, and they have also been used as additives for food industry. It is also important to note that cyclodextrins are used for improving bioactive compound production in plant cell cultures because of cyclodextrins ability as “hosts” of bioactive compounds favoring their accumulation in aqueous media. In fact, the treatment of plant cell cultures with cyclodextrins and their derivatives increases the production of secondary metabolites such as resveratrol, ajmalicine, serpentine, lutein, arachidin, among other antioxidant compounds, enhancing the capability of plant in vitro cultures to produce high levels of different bioactive compounds which have beneficial properties for human health. In addition, metabolomic, transcriptomic, and proteomic studies have been carried out on both control and cyclodextrins-treated cell cultures offering important clues about how cyclodextrins are capable of substantially increasing the production of bioactive compounds in plant in vitro cultures. This review focuses on the effect of cyclodextrins on both bioactive compound production and their accumulation, which could be of great interest for chemical and pharmaceutical industries.

Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells.

Human rhinoviruses (HRVs) are associated with acute exacerbations in patients with chronic obstructive pulmonary disease (COPD) and asthma, which are accompanied by mucus hypersecretion. Whereas, various studies have shown that HRVs increase epithelial mucin production and thus may directly contribute to mucus hypersecretion. The effects of drugs used in the treatment of COPD and asthma on HRV-induced mucin production in epithelial cell cultures have not been studied. In the present study, we assessed effects of HRVs on mucin production and secretion in well-differentiated primary human bronchial epithelial cells (PBEC) and studied the effect of the inhaled corticosteroid fluticasone propionate and the long-acting muscarinic antagonist tiotropium bromide on this process. Differentiated PBEC that were cultured at the air-liquid interface (ALI-PBEC) were infected with HRV-A16 and HRV-1B. Quantitative PCR, immunofluorescence staining, ELISA, periodic acid-Schiff (PAS) staining and immunostaining assays were used to assess the effects of HRV infection. Here we demonstrate that both HRV-A16 and HRV-1B increased mucin (MUC5AC and MUC5B) gene expression and protein release. When exploring this in more detail in HRV-A16-infected epithelial cells, mucin expression was found to be accompanied by increases in expression of SAM-pointed domain-containing Ets-like factor (SPDEF) and SPDEF-regulated genes known to be involved in the regulation of mucin production. We also found that pre-treatment with the purinergic P2R antagonist suramin inhibits HRV-enhanced MUC5AC expression and protein release, implicating involvement of purinergic signaling by extracellular ATP. We furthermore found that both fluticasone and tiotropium decreased HRV-induced mucin production without affecting viral replication, and obtained evidence to suggest that the inhibitory effect of fluticasone involved modulation of SPDEF-regulated genes and extracellular ATP release. These data show that both tiotropium and fluticasone inhibit HRV-induced epithelial mucin production independent of viral clearance, and thus provide insight into the mechanisms underlying beneficial effects of tiotropium and fluticasone in the treatment of COPD, asthma and accompanying exacerbations in these patients. Furthermore, our findings provide additional insight into the mechanisms by which HRV increases epithelial mucin production.

Characterization of Bispecific Antibody Production in Cell Cultures by Unique Mixed Mode Size Exclusion Chromatography.

Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.

The comparative analysis of stilbene production in cell cultures of two East Asian grape species Vitis amurensis Rupr. and Vitis coignetiae Pulliat

Two East Asian grape species Vitis amurensis and Vitis coignetiae have been investigated for their increased cold and drought tolerance and high level of resistance to infections. Nowadays, the grape species attract attention due to their capability to produce a group of valuable plant secondary metabolites named stilbenes. The basic stilbene, t-resveratrol, is known to demonstrate a high bioactivity and is synthesized by stilbene synthase (STS) in grape cells. In order to find the suitable biological model for t-resveratrol production, 12 cell culture lines derived from the leaf, petiole and stem explants of the East Asian grape species were tested for their ability to produce t-resveratrol and other stilbenes. The comparative analysis showed that the V. coignetiae cell culture lines were the most productive in terms of stilbene content. This observation was confirmed by the increased level of STS expression in V. coignetiae cells in comparison to V. amurensis. The expression analysis of the genes encoding transcription factors potentially involved in regulation of STS genes revealed the significant species-specific differences, suggesting the molecular basis underlying the observed differences in stilbene biosynthetic potential of V. amurensis and V. coignetiae. The comparative analysis revealed the greater stilbene production potential for cells of V. coignetiae due to enhanced STS expression determinated by Myb1/15, Myb40 and Myb60 transcription factors.

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kL a ratio as a direct indicator of gas stripping conditions.

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO2 ) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO2 was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the kL a values of O2 (kL a[O2 ]) and CO2 [kL a(CO2 )] in the SS bioreactors as a scale-up factor for dCO2 reduction. The kL a ratios [kL a(CO2 )/kL a(O2 )] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the kL a ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO2 trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This kL a ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The kL a ratios for the SUBs were evaluated and conditions that provided kL a ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the kL a ratio is a powerful scale-up factor useful to control dCO2 during fed-batch cultures.

Elemental metal variance in cell culture raw materials for process risk profiling.

Elemental metals are critical raw material attributes which can impact cell culture performance and associated therapeutic protein product quality profiles. Metals such as copper and manganese act as cofactors and reagents for numerous metabolic pathways which govern cell growth, protein expression, and glycosylation, thus mandating elemental monitoring. The growing complexity of modern cell culture media formulations adds additional opportunities for elemental variance and its associated impact risks. This article describes an analytical technique applying inductively coupled plasma mass spectrometry to characterize a list of common raw materials and media powders used in mammalian cell culture and therapeutic protein production. We aim to describe a method qualification approach suitable for biopharmaceutical raw materials. Furthermore, we present detailed profiles of many common raw materials and discuss trends in raw material subtypes. Finally, a case study demonstrating the impact of an unexpected source of raw material variation is presented along with recommendations for raw material elemental risk profiling and control.