Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

Enhanced production of alpha-amylase in fed-batch cultures of Bacillus subtilis TN106[pAT5

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc.     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

动物细胞流加培养的技术现状和发展趋势

流加培养是当前重组蛋白生产的主流培养模式。流加式操作主要是根据细胞对营养物质的不断消耗和需求,设计连续或半连续的流加浓缩营养物,使细胞持续高密度的生长,提高单位反应器体积内目的蛋白产量,从而达到高效生产的目的。流加培养工艺的关键技术主要包培养基的优化设计、流加策略的选择及优化、细胞代谢的调控。     

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

Metabolically optimised BHK cell fed-batch cultures

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.     

Enhanced growth of Sf-9 cells to a maximum density of 5.2 x 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture

Significant improvement in cell growth and protein production has been achieved in Sf-9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bédard et al., 1994; Chan et al., 1998). The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner. Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 x 10(7) cells/mL of Sf-9 cells in a 3.5 L bioreactor. Production of recombinant protein beta-galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 x 10(6)cells/mL. Specific productivity could be maintained at cell densities as high as 14 x 10(6) cells/mL. The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added. On-line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities.     

Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures

Transient continuous cultures constitute a means to speed up strain characterization, by avoiding the need for many time-consuming steady-state experiments. In this study, mixed substrate growth on glycerol and methanol of a Pichia pastoris strain expressing and secreting recombinant avidin was characterized quantitatively by performing a nutrient gradient with linear increase of the methanol fraction in the feed medium from 0.5 to 0.93 C-mol C-mol(-1) at a dilution rate of 0.06 h(-1). The influence of the methanol fraction in the feed medium on recombinant avidin productivity and on specific alcohol oxidase activity were also examined. Results showed that, compared with cultures on methanol as sole carbon source, the specific recombinant avidin production rate was the same provided the methanol fraction in the feed medium was higher than 0.6 C-mol C-mol(-1). The volumetric avidin production rate was even 1.1-fold higher with a methanol fraction in the feed medium of 0.62 C-mol C-mol(-1) as a result of the higher biomass yield on mixed substrate growth compared with methanol alone. Moreover, since heat production and oxygen uptake rates are lower during mixed substrate growth on glycerol and methanol, mixed substrate cultures present technical advantages for the performance of high cell density P. pastoris cultures. Results obtained in a high cell density fed-batch culture with a mixed feed of 0.65 C-mol C-mol(-1) methanol and 0.35 C-mol C-mol(-1) glycerol were in agreement with results obtained during the transient nutrient gradient.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg).

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.     

Production of toluene cis-glycol using recombinant Escherichia coli strains in glucose-limited fed batch culture

Production of toluene cis-glycol (TCG) was investigated using recombinant Escherichia coli strains that express toluene dioxygenase under the tac promoter. E. coli TG2 was selected as the host for the recombinant plasmid, pTAC365, because the TCG yield was 64% higher than with strain JM105 as the host. By using fed-batch culture, TCG production could be improved by 4.4-fold compared with batch cultures, with a toluene vapor feed. A further improvement of 1.2-fold was obtained by using a two-liquid phase culture system, but the improvement was 1.6-fold when the feed rate of toluene vapor was reduced by 75%. When the period of growth in fed-batch culture was extended and the reduced vapor feed was used, the maximum TCG concentration increased to 4 g/l, an overall improvement of 10-fold compared with the batch culture system used initially. However, product consumption was observed during the late stages of fed-batch growth and in stationary phase, resulting in the formation of o-cresol, 3-methylcatechol and benzyl alcohol. We conclude that the key to optimizing TCG production by recombinants is to prolong growth of the cells to ensure sustained growth-linked product formation, and to optimize the supply of the toxic substrate, toluene, but further work is needed to eliminate by-product formation.     

Process development for an inducible rituximab-expressing Chinese hamster ovary cell line

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 ⁶ cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 ⁶ cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019     

Experimental and modelling study of different process modes for retroviral production in a fixed bed reactor

Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however, low-vector concentration in batch cultures and high temperature-dependent decay do limit sufficiently large-scale production. To overcome these obstacles, the kinetic relations of cell growth and vector formation in different culture modes need to be understood. Effective optimisation of process modes is needed to achieve sufficient yields. Experimental and modelling studies were carried out in order to analyse the impact of different process modes such as perfusion, perfused fed-batch or repeated-batch on vector titer and productivity. Retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1) were produced using a 200 ml fixed bed reactor for high cell density cultivation on macroporous carriers. After starting the cultivation in batch mode, the reactor was either run in perfusion, perfused fed-batch or repeated-batch. A mathematical model of the bioreaction was developed on the basis of experimental data measured in culture dishes. The ability of the model to describe all different process modes of fixed-bed cultivation without additional fitting of the parameters was proven by three long-term cultivations for more than 400 h. The results of optimisation with the aid of the model, leads to the conclusion that perfusion with optimised harvest cycles and fed flows, result in a higher yield in comparison to batch or fed batch culture.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

Insights into adenoviral vector production kinetics in acoustic filter-based perfusion cultures

One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.     

Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).     

Modeling of the pyruvate production with Escherichia coli: comparison of mechanistic and neural networks-based models

Three different models: the unstructured mechanistic black-box model, the input–output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249–258 (2004); Zelić et al. Eng Life Sci 3:299–305 (2003); Zelić et al Biotechnol Bioeng 85:638–646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies

A fed-batch cell culture process was developed that has general applicability to all evaluated Sp2/0 (n = 8) and NS0 (n = 1) antibody-producing cell lines. The two key elements of this generic process were a protein-free concentrated feed medium, and a robust, metabolically responsive feeding strategy based on the off-line measurement of glucose. The fed-batch process was shown to perform equivalently at the 15 L development scale and 750 L manufacturing scale. Compared to batch cultures, the fed-batch process yielded a 4. 3 fold increase in the average integral of viable cell concentration and a 1.7 fold increase in average specific antibody production rate, equivalent to a 7.6 fold increase in average final antibody concentration. The highest producing cell line reached a peak viable cell concentration of 1.0 x 10(7) cell mL(-1) and a final antibody concentration of 750 mg L(-1) in a 10 day process. For all lines evaluated, reducing bioreactor pH set point from 7.2 to 7.0 resulted in an additional 2.4 fold increase in average final antibody concentration. The optimized fed-batch process consistently yielded a volumetric productivity exceeding 50 mg L(-1) day(-1). This generic, high-yielding fed-batch process significantly decreased development time, and increased manufacturing efficiency, thereby facilitating the clinical evaluation of numerous recombinant antibodies.     

Bcl-x(L) overexpression delays the onset of autophagy and apoptosis in hyperosmotic recombinant Chinese hamster ovary cell cultures

Hyperosmolality in recombinant Chinese hamster ovary (rCHO) cell cultures induces autophagy and apoptosis. To investigate the effect of Bcl-x_L overexpression on autophagy and apoptosis in hyperosmotic rCHO cell cultures, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x_L overexpression was subjected to hyperosmolality resulting from NaCl addition in a batch culture and nutrient supplementation in a fed-batch culture. In the batch culture, Bcl-x_L overexpression suppressed apoptosis, as evidenced by a decreased amount of cleaved caspase-7 and PARP. Concurrently, Bcl-x_L overexpression also delayed autophagy, as indicated by reduced LC3 conversion, from LC3-I to LC3-II. As a result, the cell viability and EPO production were improved by Bcl-x_L overexpression. In the fed-batch culture, the simultaneous application of Bcl-x_L overexpression and nutrient feeding increased the culture longevity and maximum EPO concentration. Taken together, Bcl-x_L overexpression delayed autophagy and apoptosis in hyperosmotic rCHO cell cultures, resulting in increased EPO production.