The role of vitamins and amino acids on hybridoma growth and monoclonal antibody production

A balanced supplementation method was applied to develop a serum and protein- free medium supporting hybridoma cell batch culture. The aim was to improve systematically the initial formulation of the medium to prevent limitations due to unbalanced concentrations of vitamins and amino acids. In a first step, supplementation of the basal formulation with 13 amino acids, led to an increase of the specific IgA production rate from 0.60 to 1.07 pg cell⁻¹ h⁻¹. The specific growth rate remained unchanged, but the supplementation enabled maintenance of high cell viability during the stationary phase of batch cultures for some 70 h. Since IgA production was not growth- related, this resulted in an approximately4-fold increase in the final IgA concentration, from 26.6 to 100.2 mgl⁻¹. In a second step, the liposoluble vitamins E and K₃ were added to the medium formulation. Although this induced a slightly higher maximal cell concentration, it was followed by a sharp decline phase with the specific IgA production rate falling to 0.47 pg cell⁻¹ h⁻¹. However, by applying a second cycle of balanced supplementation with amino acids this decline phase could be reduced and a high cell viability maintained for over 300 h of culture. In this vitamin- and amino acid- supplemented medium, the specific IgA production rate reached a value of 1.10 pg cell⁻¹h⁻¹ with a final IgA concentration of 129.8 mgl⁻¹. The latter represents an increase of approximately5-fold compared to the non- supplemented basal medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d⁻¹) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell⁻¹) compared with cells grown by other culture methods (0.27–0.49 pg cell⁻¹). The highest cell density and cellular toxin content were 17190 cells mL⁻¹ and 1.26 pg cell⁻¹ respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

Relationship between tissue plasminogen activator production and specific growth rate in Chinese Hamster Ovary cells cultured in mannose at low temperature

Chinese hamster ovary (CHO) cells, producing human recombinant tissue plasminogen activator (tPA), were grown with mannose (5, 20 and 40 mM) instead of glucose at 31, 33 and 37°C. The highest tPA concentration (1.5 mg l⁻¹ at 144 h of cultivation) and tPA specific production rate (47 ng 10⁻⁶ cell h⁻¹) were obtained at 31°C and 40 mM mannose. Regardless of the temperature or mannose concentration used, an inverse relationship between the specific growth rate and tPA specific production rate was observed, suggesting that tPA production rate would be directly controlled by the growth rate.     

Variations in the Microcystin Production of Planktothrix rubescens (Cyanobacteria) Assessed from a Four-Year Survey of Lac du Bourget (France) and from Laboratory Experiments

Between 1999 and 2002, a routine survey of water quality in the Lac du Bourget was performed to study the dynamics and microcystin (MC) production of Planktothrix rubescens. Using liquid chromatography coupled to diode array detection and mass spectrometry, we found that two main variants ([d-Asp³] and [d-Asp³, Dhb⁷] microcystin-RR) were produced. The proportion of these two variants was not influenced by the depth or season of sampling. Expressed in microcystin-LR equivalents, high microcystin concentrations were recorded from August to December each year, reaching values of up to 6.7 μg L⁻¹. A significant correlation was found between the microcystin cell content and the cell densities of P. rubescens. Cellular quotas of microcystins ranged from 0.1 to 0.3 pg cell⁻¹. Simultaneously, laboratory experiments were performed on a strain of P. rubescens isolated from the lake to assess the potential impact of various P–PO₄³⁻ concentrations on intra- and extracellular microcystin production. Unlike natural populations, this strain only produced [d-Asp³] MC-RR. The intracellular microcystin content was similarly correlated to the cell density, but the cellular quota was slightly higher (0.3–0.7 pg cell⁻¹) than in the natural population. Again, as in the natural population, a linear relationship was found between growth rate and microcystin production rate. These findings support the hypothesis that environmental factors, such as phosphate concentrations, have no direct impact on microcystin production by P. rubescens, but act indirectly by affecting growth rate.     

Nitrogen and phosphorus utilization in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> isolated from Laguna de Bay,Philippines

Phytoplankton supports fisheries and aquaculture production. Its vital role as food for aquatic animals, like mollusks, shrimp, and fish cannot be overemphasized. Because of its contribution as a food source for fish, the growth kinetics of Microcystis aeruginosa, a dominant cyanobacterium in the lake, was studied. The regular occurrence of M. aeruginosa is experienced during the months of May to July or from September to November in Laguna de Bay, the largest freshwater lake in the Philippines. M. aeruginosa was collected from Laguna de Bay, isolated, and established in axenic conditions. Data on the growth kinetic parameters for nitrate-nitrogen and phosphate-phosphorus utilization by M. aeruginosa gave the following values: half-saturation constant (K _s ), 0.530 mg N. L⁻¹ and 0.024 mg P. L⁻¹ respectively; maximum growth rate (μ _max ), 0.671. d⁻¹ and 0.668. d⁻¹ respectively; maximum cell yield, 6.5 and 6.54 log, cells. ml⁻¹ respectively; nutrient level for saturated growth yield, 8.71 mg N. L⁻¹ and 0.22 mg P. L⁻¹ respectively; and minimum cell quota (Q ₀ ), 2.82 pg N. cell⁻¹ and 0.064 pg P. cell⁻¹ respectively. The low K _s value and high maximum growth rate (μ _max ) for phosphorus by M. aeruginosa would suggest a high efficiency of phosphorus utilization. On the other hand, the high K _s value for nitrogen indicated a low rate of uptake for this nutrient.     

Adenovirus vector production using low-multiplicity infection of 293 cells

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001–0.1 transductional unit (TU) cell⁻¹. The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell⁻¹), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 × 10⁵ cells cm⁻³ were infected with Ad EGFP at a low MOI of 0.001 TU cell⁻¹, the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell⁻¹) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.     

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus–insect cell expression system

A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O₂ mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 10⁶ cells ml⁻¹) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 10⁶ cells ml⁻¹ were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell⁻¹, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L⁻¹ of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 10⁶ cells ml⁻¹. Received 09 December 1996/ Accepted in revised form 13 April 1997     

Functional Characterization of the Microbial Community in Geothermally Heated Marine Sediments

The microbial population of geothermally heated sediments in a shallow bay of Vulcano Island (Italy) was characterized with respect to metabolic activities and the putatively catalyzing hyperthermophiles. Site-specific anoxic culturing media, most of which were amended with combinations of electron donors (glucose or carboxylic acids) and acceptors (sulfate), were used for selective enrichment of metabolically defined subpopulations. The mostly archaeal chemoautotrophs produced formate at rates of 3.25 and 0.46 fmol cell⁻¹ day⁻¹ with and without sulfate, respectively. The glucose fermenting heterotrophs produced acetate (18 fmol cell⁻¹ day⁻¹) and lactate (2.6 fmol cell⁻¹ day⁻¹) and were identified as predominantly Thermus sp. and coccoid archaea. These archaeal cells also metabolized lactate (5.6 fmol cell⁻¹ day⁻¹), but neither formate nor acetate. The heterotrophic culture enriched on formate/acetate/propionate/sulfate utilized mainly formate (27 fmol cell⁻¹ day⁻¹) and lactate (89–195 fmol cell⁻¹ day⁻¹), and consumed sulfate (38–68 fmol cell⁻¹ day⁻¹). These formate or lactate consuming sulfate reducers were dominated by Archaeoglobales (7% in situ) and unidentified Archaea. The in situ benthic community comprised 15% Crenarchaeota, a significant group only in the autotrophic cultures, and 3% Thermus sp., the putatively predominant group involved in fermentative metabolism. The role of Thermoccales (4% in situ) remained undisclosed in our experiments. This first comprehensive data set established plausible links between several groups of hyperthermophiles in shallow marine hydrothermal systems, their metabolic function within the benthic microbial community, and biogeochemical turnover rates.     

Changes in the cell composition of the marine microalga, Nannochloropsis gaditana, during a light:dark cycle

Nannochloropsis gaditana was grown in semicontinuous culture with a circadian light:dark cycle in a flat-panel photobioreactor. The microalga had a maximal protein content (3 pg cell^–1) after 6 h light and then only storage compounds were accumulated that were consumed during the dark phase. Carbohydrates reached their maximum value after 8 h (0.8 pg cell^–1) and lipids after 12 h light (2.5 pg cell^–1). The results demonstrated that young or adult microalgae might be obtained according to the time of day.     

Effects of reduced diameter of bag cultures on content of essential fatty acids and cell density in a continuous algal production system

Cell density and fatty acid (FA) content of Pavlova lutheri and Chaetoceros muelleri were analysed in a continuous algal production system (250-L bags) with reduced diameter. The cell density and FA content and composition in the algal production system were determined in replicate bags over a period of 5 weeks. The results showed that the cell density and essential FAs increased during the experiment for both species. After 5 weeks the mean cell numbers had increased to 6.0 ± 0.3 × 10⁶ cells mL⁻¹ in the P. lutheri bags and 6.0 ± 0.4 × 10⁶ cells mL⁻¹ in the C. muelleri bags. The content of total FAs increased significantly (p < 0.05) in all of the bags during the experiment. At the end of the experiment the mean total FA content were 2.7 ± 0.3 pg cell⁻¹ in the P. lutheri bags and 1.8 ± 0.1 pg cell⁻¹ in the C. muelleri bags. Maximum total FA content registered was 3.0 pg cell⁻¹ in one of the P. lutheri bags. The content of the essential FAs (ARA, EPA, DHA) increased over time in both of the species. At the end of the experiment the content of EPA (0.6 ± 0.1 pg cell⁻¹) and DHA (0.3 ± 0.0 pg cell⁻¹) were highest in the P. lutheri bags, while ARA (0.1 ± 0.0 pg cell⁻¹) was highest in C. muelleri. EPA and DHA constituted 22% and 11%, respectively, of total FA content in P. lutheri, while ARA constituted 6% of total FA content in C. muelleri. The results from this experiment indicate that flagellates such as P. lutheri perform better in narrow bags with improved light conditions, while diatoms like C. muelleri perform better in wider bags under light limitation. Implications for bivalve hatcheries are discussed.     

The long-term culture of the coccolithophore Pleurochrysis carterae (Haptophyta) in outdoor raceway ponds

The calcareous marine haptophyte algae, the coccolithophorids, are of global environmental significance because of the impact of their blooms on the carbon cycle. The coccolithophorid, Pleurochrysis carterae was grown semi-continuously in paddlewheel-driven outdoor raceway ponds over a period of 13 months in Perth, Western Australia. The mean total dry weight productivity of P. carterae was 0.19 g.L⁻¹.d⁻¹ with cell lipid and CaCO₃ contents of up to 33% and 10% of dry weight respectively, equivalent to an annual total biomass productivity of about 60 t.ha⁻¹.y⁻¹ and 21.9 t.ha⁻¹.y⁻¹ total lipid and 5.5 t.ha⁻¹.y⁻¹ total calcium carbonate production. Throughout the culture period there was little protozoan contamination or contamination by other algae. The pH of the growth medium increased to pH 11 during the day and was found to be a useful variable for monitoring the state of the culture. A comparison of the growth of P. carterae and Dunaliella salina in the raceway ponds showed no significant differences between these two species with regard to areal total dry weight productivity and lipid content.     

Stability validation of seeding cell control parameters in large-scale hybridoma cell culture

Stability and reproducibility of seeding cell performance in large-scale hybridoma cell culture has been reported by controlling only initial cell seeding density. The aim of the current study was to integrate multiple seeding cell control parameters to maintain stable and consistent cell physiological status for HAb18 cell expansion. Three parameters and their ranges were investigated, including initial cell seeding density in the range of 0.075–0.5×10⁶ cells ml⁻¹, “timepost” after cell passage between 8 and 36 h, and duration of subculture up to 6 months after cell revival. Cell performance was tested at the 1 L, 5 L, and 75 L scales. Desirable performance was found within the following parameter ranges: initial cell seeding density of 0.1–0.3×10⁶ cells ml⁻¹, “timepost” after cell passage between 14 and 22 h, and duration of subculture within 3 months of cell revival. Our results showed that cell growth rate and antibody productivity of three batches at 1 L, 5 L, and 75 L scale were found to be stably maintained within a range of 0.036–0.047 h⁻¹ and 0.577–0.747 pg cell⁻¹ h⁻¹, with the positivity rate of antigen-binding activity within 97–99.75%, and the intensity of fluorescence around 200. This study may provide a simple but effective method to maintain seeding cell physiological status stable and consistent by combining seeding cell control parameters.     

Vat incubator with immersion core illumination — a new,inexpensive setup for mass phytoplankton culture

One of the shortcomings in studies of bivalve grazing has been the difficulty of culturing and making available sufficient quantities of algae. This was overcome using a 2501 capacity vat incubator with immersion core illumination (VIICI) in connection with experiments involving the diatom Nitzschia pungens f. multiseries, which produces domoic acid, the cause of amnesic shellfish poisoning. Nitzschia cultures grown in this incubator yielded maximum cell concentrations of 158–166 × 10⁶ cells 1⁻¹, a peak intracellular domoic acid level of 2.0 pg cell⁻¹ and a maximum division rate of 0.3 d ⁻¹. The VIICI design is ideally suited for laboratory mass culture of phytoplankton, and has potential for wide application in phycotoxin, toxicological and environmental research, as well as for aquaculture.     

Growth of Amoebae and Flagellates on Bacteria Deposited on Filters

Abstract Artificial bacterial biofilms were formed by making microwave-irradiated, dual-radioisotope-labelled Vibrio bacteria adhere to 0.4 μm pore size filters with albumin. The rate of release of ³H from thymidine label in these bacteria into the surrounding seawater when protozoa were incubated with the biofilm indicated the predator's grazing rate, and the rate of accumulation of ¹⁴C in the predators from leucine label in the bacteria indicated the assimilation rate of the protozoa. The amoeba Vanella septentrionalis consumed about 60% of the available bacteria between the 5th and 15th days of incubation with a gross growth efficiency of 22 ± 6%, compared with about 75% consumption at 29 ± 8% efficiency for the surface-feeding flagellate Caecitellus parvulus, and about 55% consumption at 16 ± 5% efficiency for the suspension-feeding flagellate Pteridomonas danica. As a result of their grazing and metabolism these protozoa regenerated about 70–85% of the nutrients present in their food and released these nutrients in the immediate vicinity of the bacterial biofilm. The biomass of the amoeba Vanella was calculated to be 166 pg protein cell⁻¹ during maximum growth and 93 pg protein cell⁻¹ in the stationary phase. Received: 3 August 1998; Accepted: 20 November 1998     

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of ∼5 × 10⁶ cells mL^-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell^-1 day^-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized (∼40 mg L^-1) at 0.2 nL cell^-1 day^-1. The volumetric protein productivity (∼60 mg L^-1 day^-1 was maximized above 0.3 nL cell^-1 day^-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Photosynthetic characteristics of Dunaliella salina (Chlorophyceae,Dunaliellales) in relation to β-carotene content

Photosynthetic characteristics of Dunaliella salina with high (red form) and low β-carotene (green form) concentrations were studied. D. salina growing in brine saltworks exhibited a high level of β-carotene (15 pg cell⁻¹). The rate of oxygen evolution as a function of irradiance was higher in the red than in the green form (on chlorophyll basis). Photosynthetic inhibition of the green form was observed above 500 μmol m⁻² s⁻¹. The red form appeared more resistant to high irradiance and no inhibition in O₂ evolution was observed up 2000 μmol m⁻² s⁻¹. However, when these results are expressed on a cell number basis the rate of oxygen evolution was significantly higher in the green form. Carbonic anhydrase (CA) activity (total, soluble, membrane bound) was found in red and green forms. CA was higher in the red form on a chlorophyll basis, but lower if expressed on a protein basis. The light dependent rate of oxygen evolution and photoinhibition depends on the concentration of β-carotene in D. salina cells.     

Phytoplankton primary production in a mesotrophic pond in sub-tropical Bangladesh

Annual gross primary productivity in mesotrophic Shahidullah Hall pond (Dhaka, Bangladesh) was 1383.35 g C m⁻² y⁻¹ (arithmetic mean). Daily primary productivity (between 1.6 and 6.8 g C m⁻² d⁻¹ was correlated with chlorophylla, day length and dissolved silica. Chlorophylla related significantly withk, incident light, SRP, alkalinity and conductivity. A negative correlation existed between biomass and rainfall. Productivity, biomass, conductivity, alkalinity, and SRP increased after mid-winter.k, I _k andZ _eu varied according seasonally.P _max related directly with temperature. Seasonal variation of ∝ ^B was 0.0049–0.0258 mg C (mg chla mmol PAR)⁻¹ m⁻². Q₁₀ was 2.12, community respiration 1334.99 g C m⁻² y⁻¹, and the underwater light climate 186.43μE m⁻² s⁻¹.     

The relative importance of tryptophan-dependent and tryptophan-independent biosynthesis of indole-3-acetic acid in tobacco during vegetative growth

A quantitative study of indole-3-acetic acid (IAA) turnover, and the contribution of tryptophan-dependent and tryptophan-independent IAA-biosynthesis pathways, was carried out using protoplast preparations and shoot apices obtained from wild-type and transgenic, IAA-overproducing tobacco (Nicotiana tabacum L.) plants, during a phase of growth when the level of endogenous IAA was stable. Based on the rate of disappearance of [¹³C₆]IAA, the half-life of the IAA pool was calculated to be 1.1 h in wild-type protoplasts and 0.8 h in protoplasts from the IAA-overproducing line, corresponding to metabolic rates of 59 and 160 pg IAA (μg Chl)⁻¹ h⁻¹, respectively. The rate of conversion of tryptophan to IAA was 15 pg IAA (μg Chl)⁻¹ h⁻¹ in wild-type protoplasts and 101 pg IAA (μg Chl)⁻¹ h⁻¹ in protoplasts from IAA-overproducing plants. In both instances, IAA was metabolised more rapidly than it was synthesised from tryptophan. As the endogenous IAA pools were in a steady state, these findings indicate that IAA biosynthesis via the tryptophan-independent pathway was 44 pg IAA (μg Chl)⁻¹ h⁻¹ and 59 pg IAA (μg Chl)⁻¹ h⁻¹, respectively, in the wild-type and transformed protoplast preparations. In a parallel study with apical shoot tissue, the presumed site of IAA biosynthesis, the rate of tryptophan-dependent IAA biosynthesis exceeded the rate of metabolism of [¹³C₆]IAA despite the steady state of the endogenous IAA pool. The most likely explanation for this anomaly is that, unlike the protoplast system, injection of substrates into the apical tissues did not result in uniform distribution of label, and that at least some of the [²H₅]tryptophan was metabolised in compartments not normally active in IAA biosynthesis. This demonstrates the importance of using experimental systems where labelling of the precursor pool can be strictly controlled. Received: 18 January 2000 / Accepted 24 February 2000