High density culture of HeLa cells in a CelliGen perfusion system

A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor. Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems. This system successfully propagated HeLa cells to over 11 million viable cells per milliliter. Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate. Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells. Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor.     

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.     

Application of in situ product-removal techniques to biocatalytic processes.

Biocatalytic processes for the manufacture of small, highly functionalized molecules frequently have limited productivity. A common reason for this is the presence of the reaction products that can cause inhibitory or toxic effects (making poor use of the enzyme) or promote unfavourable equilibria (giving low conversions). In each case, the product needs to be removed as soon as it is formed in order to overcome these constraints and hence increase the productivity of the biocatalytic process. Here, we review the need for in situ product removal and the process research required for its implementation.     

Novel differential and confirmation plating media for Shiga toxin-producing Escherichia coli serotypes O26, O103, O111, O145 and sorbitol-positive and -negative O157

This study reports two novel selective differential media. A first differential medium can be applied in methods for the isolation of non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes (O26, O103, O111 and O145) from food or faeces. A second differential medium was designed for both sorbitol-positive and -negative O157 STEC strains. Selective differential media are based on a chromogenic compound to signal beta-galactosidase activity and one or more fermentative carbon sources. The chromogenic marker and carbohydrates were combined with a pH indicator and several inhibitory components, which resulted in highly specific differentiation media. Consecutive use of a serotype-dependent choice of confirmation media resulted in a very low incidence of false-positive isolates when comparing clinical STEC strains with a collection of commensal E. coli strains.     

Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells

In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel^® disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (2.5×10⁷ cell ml⁻¹ of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by −25% and −50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a −25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a −30% loss of productivity was obtained when the medium perfusion rate was further reduced to −50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of −25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase – provided that the product is not sensitive to residence time – with the benefits of reduced cost of goods and to simplify manufacturing operations.     

Utilization of mustard waste isolates for improved production of astaxanthin by Xanthophyllomyces dendrorhous

Astaxanthin production in the wild strain Xanthophyllomyces dendrorhous TISTR 5730 was investigated using different mustard waste media, including mustard waste residue extract (MRE), mustard waste residue hydrolysate (MRH), mustard waste precipitated extract (MPE), and mustard waste precipitated hydrolysate (MPH). The growth of X. dendrorhous and the production of astaxanthin were dependent on the type and initial concentrations of mustard waste media. The MPH medium was the best substrate resulting in yields of biomass and astaxanthin of 19.6 g/L and 25.8 mg/L, respectively, under optimal conditions. MPH medium improved astaxanthin production 11-fold compared to the commonly used commercial yeast malt medium, and 1.3–2.1-fold compared to other mustard waste media.     

Sugar degradation during autoclaving: Effects of duration and solution volume on breakdown of glucose

As the autoclaving of a sugar together with other nutrient components enhances its degradation with the associated formation of toxic products, it is advisable to autoclave it separately from other medium components. In such cases, and to prepare growth media of consistent quality, it may also be necessary to adjust the duration of autoclaving according to the volume of the sugar solution. The results of an attempt to fine-adjust the autoclaving procedure are presented.
Glucose, used as model sugar in this investigation, was autoclaved for 20,32 or 44 min in volumes of 0.25. 1.2 and 5 1. The rise in temperature of the solutions was monitored by thermocouples. Glucose degradation was estimated by the rate of cyanide-initiated oxygen consumption. The rates were found to be inversely proportional to the volumes in a semilogarithmic plot. The T-t (temperature-time) value, the area under the solution temperature vs time curve, was found to be an independent variable from which the extent of glucose degradation could be deduced. For a chosen level of thermodegradation of glucose, the duration of autoclaving for solutions of different volumes could be determined graphically from a T-t value vs time plot.     

Peptone,a low-cost growth-promoting nutrient for intensive animal cell culture

The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×10⁷ cells ml^–1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

Continuous medium perfusion leads to long-term cell viability and oxygen production in high-density photobioreactors

Summary A light-emitting diode-based photobioreactor (LED-based PBR) operated in a continuous perfusion mode with a perfusion rate of 3 to 6 reactor volumes a day supports high-density algal cultures, of cell concentrations up to 4·10⁹ cells/mL, or 25 g/L. The oxygen production rate at its peak was 13 to 15 mmol/(L·h). Continuous medium perfusion allowed for long-term stable oxygen production, while oxygen production in batch mode ceased when stationary phase was reached.     

Product enhancement and recovery from transformed root cultures of Nicotiana glauca

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.     

Development of novel Alicyclobacillus spp. isolation medium

AIM: To develop a new isolation medium with higher recovery rates of Alicyclobacillus spp. METHODS AND RESULTS: SK agar was developed with optimized incubation temperature, pH, acidulant, Tween 80 concentration and divalent cation addition. Results indicate that detection of Alicyclobacillus spp. by SK agar was significantly higher (P > 0.05) than those obtained by K agar, orange serum agar, and potato dextrose agar. CONCLUSIONS: Current media used for Alicyclobacillus spp. isolation still resulted in high numbers of false negative products. The sensitivity of SK agar to Alicyclobacillus spp. allows detection of low numbers of Alicyclobacillus spp. and also provides a more higher isolation results compared with currently used media. SIGNIFICANCE AND IMPACT OF THE STUDY: SK agar will be useful to the fruit juice industry to obtain more accurate numbers of contaminant Alicyclobacillus spp. With this media, false negative samples can be reduced, and the likelihood of exported products being rejected can be greatly reduced.     

Perfusion bioreactors for the production of recombinant proteins in insect cells

Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.     

Production of rosmarinic acid in high density perfusion cultures ofAnchusa officinalis using a high sugar medium

Summary The most direct approach to enhancing the volumetric yield of secondary metabolites in plant tissue cultures is to operate the culture under high cell density. In this study, a cell suspension ofAnchusa officinalis was cultivated using a semi-continuous perfusion technique, i.e. batch cultivation with intermittent medium exchange. Using a perfusion medium containing sucrose concentration which was two times that in the normal growth medium, the final cell density and the final product concentration were increased by more than 2-fold compared with a batch culture without medium exchange. The high cell density obtained from the semi-continuous perfusion culture can be explained by the prevention of nutrient depletion, removal of toxic by-products, as well as the control of cell size by virtue of the high sugar medium osmolarity.     

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

Tryptophan oxidation catabolite,N‐formylkynurenine,in photo degraded cell culture medium results in reduced cell culture performance

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC‐MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N‐formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:74–82, 2016     

Rapid production of Pediococcus halophilus salt-tolerant cells by a cultivation method employing gradual increases in NaCl concentration using a fermentor with a microfiltration module

A new fermentation system employing gradual increases in the NaCl concentration of the culture medium was developed for the rapid production of Pediococcus halophilus NaCl-tolerant cells. A fermentation system with cross-flow filtration using a micro-filtration module allowed the continuous removal of inhibitory metabolites and complete recycling of the cells to the fermentor. By increasing the NaCl concentrations stepwise or linearly in the feeding media, cells of 8.48 or 9.86 g-dry weight per liter were obtained. The productivities of NaCl-tolerant cells per unit time in a cultivation with a stepwise or linear increase in NaCl concentration in the feeding media were 46 or 56-fold as high, repectively, as that in conventional batch cultivation.     

New bacterial culture medium for production of mosquito pathogenic bacilli using agro-poultry industrial wastes

A cost-effective technology has been developed to utilise bioorganic wastes as culture media, to produce mosquitocidal biopesticides, Bacillus sphaericus and B. thuringiensis serovar israelensis. The mosquitocidal spore/crystal toxins produced from the experimental medium (chicken feather waste, CFW+paddy husk waste, PHW) was higher than that of the conventional medium (Nutrient Yeast Extract Salt Medium, NYSM). The bacterial toxins produced from different media (NYSM, CFW, PHW, CFW+PHW) were bioassayed against mosquito vectors and the toxic effect was found to be significant. The use of chicken feathers and paddy husk wastes as bacterial culture media is cost-effective and economical for the production of these mosquito pathogenic bacilli.     

The role of activated charcoal in plant tissue culture

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.