Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.Institut National Polytechnique de Lorraine, ENSAIA BP 172, 2 avenue de la forêt de Haye, 54505, Vandoeuvre Cedex France     

Effects of inoculum size and age on biomass growth and paclitaxel production of elicitor-treated Taxus yunnanensis cell cultures

Suspension cultures of Taxus yunnanensis cells were inoculated with cells of different culture ages (12-24 days) at various densities [50-250 g fresh weight (fw)/l], and treated (on day 7) with a mixture of elicitors, including Ag(+), chitosan and methyl jasmonate. The biomass productivity (during the production stage) increased dramatically with inoculum size, but decreased with inoculum age over 16 days. The volumetric yield and productivity of taxol (paclitaxel) also increased with inoculum size, while the specific taxol yield (per cell) was mainly dependent on inoculum age, with an optimum of 20 days, during the early stationary phase. The highest taxol yield and productivity, 39.8 mg/l and 1.9 mg/l per day, respectively, were obtained with a 20-day-old inoculum at 200 g fw/l. Taxol excretion by the cells increased with inoculum age but decreased with inoculum size. The elicitor-induced activities of catalase (CAT) and phenylalanine ammonia-lyase (PAL) also depended mainly on inoculum age; higher PAL activity and lower CAT activity were obtained with an older inoculum, corresponding to a higher taxol yield. The results show that both inoculum size and age are important variables for taxol production, though the latter more profoundly influences elicitor-induced taxol biosynthesis of the cells. Inoculum size and age are also interrelated and should be optimized together in a two-stage culture process.     

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.     

Effect of low inoculation density in the scale-up of insect cell cultures

The scale-up of insect cell cultures and the production of baculovirus with these cultures is dependent on the inoculation density applied. The effect of applying a low inoculation on the specific growth rate and on the duration of the lag phase was tested. Three different cell lines, HzAm1, Ha2302, and Sf21 were tested in a total of five cell line/medium combinations. Growth in suspension culture was examined, and data obtained were fitted with the Gompertz equation. A significant decline in specific growth rate with decreasing inoculation density was observed in all cell line/medium combinations, except for HzAm1. No critical inoculation density, below which no growth would occur, was found. In suspension culture in shake flasks, an inoculation density of 5 x 10(4) cells/mL is achievable, without severely influencing the overall growth rate. A lower inoculation density in suspension culture results in less steps in the scale-up process and might be a tool in bypassing the viral passage effect.     

Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production.

A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 10(2) to 10(5) cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 10(6) cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 10(3) cells per ml grew 30% slower than those at 10(4) or 10(5). This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.     

Modeling linear and variable growth in phosphate limited suspension cultures of Opium poppy

In examining the growth kinetics of cell suspensions of opium poppy (Papaver somniferum), the increase in biomass with time was observed to be linear over the entire batch growth period of up to 20 days. Although batch growth profiles were reproducible utilizing the same inoculum, growth rates varied tremendously when experiments were inoculated with cells from different flasks. Both of these phenomena are difficult to explain with conventional batch growth models. In a series of a experiments, phosphate was determined to be the growth-rate-limiting substrate. By expressing growth rate in terms of the intracellular reserves of phosphorus, a growth model which expresses kinetics in terms of the intracellular phosphorus contents of the cells is shown to predict both linear growth character and inoculum dependent variability in growth. The stationary phase phosphate content of seven plant suspension cultures of different plant species was found to be comparable to phosphorus levels of phosphate-starved poppy cells, which suggests that phosphate limitation may be common for plant tissue culture. The applicability of this model to other biological systems which display similar batch growth patterns when subjected to inorganic nutrient deprivation is discussed.     

Production of Choristoneura fumiferana Nucleopolyhedrovirus in C. fumiferana (CF-2C1 Cells) in a 3 Litre Bioreactor Using Serum-free Medium

The purpose of this study was to develop a cell culture process in a bioreactor for the production of a viral insecticide for the spruce budworm, Choristoneura fumiferana . Several cell lines were tested for their growth in serum-free medium suspension cultures. One cell line, CF-124T-2C1 (CF-2C1), was successfully adapted to grow in suspension cultures in SFM. Serum-free Ex-Cell 405 medium produced a much higher cell density (6.3 x 10 6 cells ml -1 ) than the Grace's medium supplemented with 10% fetal bovine serum (2.5 x 10 6 cells ml -1 ). Also, a higher yield of virus was obtained in the former medium. Ex-Cell 405, was used to study the growth of CF-2C1 cells and the production of C. fumiferana nucleopolyhedrovirus (CfMNPV) in a 3 l bioreactor. Under these conditions, a specific growth rate ( μ) of 0.027 h -1 was obtained during the exponential growth phase, and the specific carbon dioxide evolution rate, as determined by on-line measurement, was 0.9 x 10 -16 mol cell -1 s -1 and 1.78 x 10 -16 mol cell -1 s -1 during growth and infection phases, respectively. Virus production in bioreactor cultures infected at 1.3 x 10 6 cells ml -1 was consistently lower than that obtained in Erlenmeyer shake flasks. Only 26% of the cells were infected in the bioreactor compared to 44% in the shake flasks. However, a higher yield of occluded virus was obtained in the bioreactor cultures than in shake flasks. The production of occlusion bodies (OB) achieved in bioreactor cultures was 2 x 10 6 OB ml -1 .     

Growth and Enzyme Activity of Myxococcus virescens in Liquid Medium

The object of this work was to develop a method for determination of the growth of Myxocuccus virescens in liquid medium. The bacteria were grown in N III-B medium in 100 ml Kjeldahl flasks, which were fixed on a disc forming an angle of 50 degrees with the horizontal plane. The disc was rotated two full revolutions per minute. The total nitrogen content of the washed swarms, developing on the glass walls of the flasks, was used as an expression for the myxobacterial growth. After a lag the bacteria grown in total darkness had a growth phase, approximately exponential, of about 270 hours, which was followed by a steep phase of decline. When the bacteria were illuminated daily for a short period, a lag of 50–200 hours appeared in the middle of the exponential growth phase, after which a new exponential growth phase began. This second growth seemed to depend on variants insensitive to light induced lysis. The increase of enzymes, active on casein and autoclaved aerobacter cells, closely followed the first part of the growth curve. However both activities began to decrease before the growth maximum. No sign of proteolytic activity or lytic activity on autoclaved aerobacter cells could be detected after about 700 hours' incubation. In illuminated flasks it is shown that the production of yellow pigments in culture solution is sharply increased at the end of the exponential growth phase. The lytic enzymes of M. virescens seem to be extracellular, secreted during the exponential phase of growth. No activity was exhibited by washed cell swarms, even if they were sonically disintegrated.     

The production of monoclonal antibody in growth-arrested hybridomas cultivated in suspension and immobilized modes.

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.     

Ambruticin S production in amino acid rich media

Summary The antimycoticum ambruticin was produced by cultivating the myxobacterium Sorangium cellulosum in shake flasks and stirred tank reactors with 10- and 30-1 working volumes. During growth and product formation amino acid concentrations in the complex media were measured by HPLC. In the logarithmic growth phase free aspartate and glutamate were assimilated without product formation. After consumption of aspartate and glutamate, growth was limited and ambruticin production set in. The effects of biological parameters such as age, amount of inoculum, pretreatment of cells before inoculation and time of precultivation were also investigated.Offprint requests to: W.-D. Deckwer     

胀果甘草悬浮培养细胞合成甘草总黄酮

比较了胀果甘草(Glycyrrhiza inflata)悬浮细胞在逐级放大摇瓶中的生长、黄酮产量以及营养消耗过程,以便了解其放大规律。结果表明,在250和500mL摇瓶中,细胞的最大生物量、黄酮产量以及最大比生长速率没有显著性差异,但是在1L的摇瓶中,这三种参数都显著地降低,分别比250mL摇瓶中降低了27%,30%和27%。在逐级放大的摇瓶中,氮、磷、铵浓度都随着培养时间延长而逐渐降低,尽管在1L的摇瓶中磷消耗得最慢,但三种摇瓶中磷在细胞生长对数期基本都被消耗尽了。此外,硝态氮在第18天时基本被消耗完,而铵态氮在细胞收获时仍能维持在100mg/L。因此在反应器中培养时,主要的培养条件还需进一步优化。     

Conditioned medium from Listeria innocua stimulates emergence from a resting state: not a response to E. coli quorum sensing autoinducer AI-2

The lag phase of the bacterial growth curve is an important determinant in speeding the detection of pathogens. It is affected by many factors including the prevailing growth environment and inoculum size, as well as specific signal molecules. The elucidation of growth-regulating signal molecules is further facilitated by culturing cells in defined growth media. In this study, a defined medium capable of supporting growth of Listeria innocua at similar levels as obtained using a complex brain heart infusion (BHI) media was developed. Further, the effects of conditioned medium (CM) on population lag time of L. innocua was investigated using a rapid parallel approach (with an automated microtiter plate reader). Importantly, the lag phase was shortened by up to approximately 50% by the addition of CM from L. innocua cultures obtained late in the exponential phase. Finally, while L. innocua were found to secrete bacterial signaling autoinducer, AI-2, tests using Escherichia coli based CM having a 90-fold difference in AI-2 level suggested that the observed decrease in lag phase was not due to E. coli-derived AI-2 and was instead due to elements specific to L. innocua. These findings indicate secreted signal molecules may be found in CM that speed detection of L. innocua.     

A 24-microwell plate with improved mixing and scalable performance for high throughput cell cultures

Multi-well plates are widely used in high throughput drug screening, cell clone development, media design and cell culture optimization in the biotechnology industry. The reproducibility and data quality of cell cultures in multi-well plates are greatly affected by mixing, aeration, and evaporation. A novel 24-microwell plate (MWP) with static mixers for improved mixing and aeration, and gas permeable lids for reduced evaporation was developed for cell cultures. Mixing, oxygen transfer, evaporation, and cell proliferation as affected by the static mixer, shape of the well and agitation rate were studied. The static mixer improved mixing pattern and reduced cell aggregation under orbital shaking conditions. Consequently, the static mixer also improved cell proliferation with a significantly higher specific growth rate in round wells. In general, consistent growth kinetics was observed for cells cultured on the plate. Overall, the MWP improved the data quality with smaller standard deviations and better reproducibility. Furthermore, CHO cells cultured in the MWP gave similar kinetics in glucose consumption, lactate production, cell growth and viability, and antibody production in a serum-free medium to those cultured in spinner flasks, demonstrating its scalable performance and potential application in high throughput screening for cell culture process development.     

Effect of the viable cell count on the growth parameters of Leptospira cultures

The influence of the amount of live cells on the growth characteristics of 41 Leptospira pathogenic strains belonging to 4 serogroups at different stages of growth has been studied. The study has revealed that under the conditions of batch cultivation the maximum concentration of pathogenic leptospires in the inoculum decreases the duration of the lag phase and determines the highest specific growth rate characterizing the individual features of leptospires in the serogroups under study.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch     

Relationship between hybridoma growth and monoclonal antibody production.

Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during G1 and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.     

TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 10⁶ cells/ml, maximal cell densities of 16 × 10⁶ and 6 × 10⁶ cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5 × 10⁶ cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 10⁶ cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production.