Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.     

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions.

We have shown previously that recombinant NS/0 myelomas expressing sufficient amounts of E1B-19K were resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. However, no significant increase in monoclonal antibodies (MAb) was observed during the prolonged stationary phase of these batch cultures. Here, we show that E1B-19K can enhance cell survival and improve MAb productivity in high cell density perfusion culture. Typically, lymphoid cells grown under steady state in perfusion exhibit decreasing viabilities with concomitant accumulation of apoptotic cells. By modulating the ability of these cells to resist to induction of apoptosis in low nutrient environment, a 3-fold decrease in specific death rate from 0.22 day-1 for NS/0 control to 0.07 day-1 for E1B-19K cells was achieved, resulting in a significant improvement in cell viability throughout perfusion. E1B-19K cells at the perfusion plateau phase also exhibited a 3-fold reduction in specific growth rate concomitant with a lower percentage of S and higher percentage of G1 phase cells. This was associated with a 40% decrease in specific oxygen consumption rate, likely related to a reduction in the specific consumption rates of limiting nutrient(s). Expression of E1B-19K consequently had a significant impact on the steady-state viable cell density, allowing maintenance of 11.5 x 10(6) E1B-19K cells/mL versus 5.9 x 10(6) control NS/0 cells/mL for the same amount of fresh medium brought into the system (half a volume per day). Whereas MAb concentrations found in perfusion culture of control NS/0 myelomas were almost 3-fold higher than those found in batch culture; in the case of E1B-19K-expressing myelomas, the MAb concentration in perfusion was more than 7-fold higher than in batch. This was attributable to the 2-fold increase in viable cell plateau and to a 40% increase in the perfusion to batch ratio of specific MAb productivity (2.2-fold for E1B-19K myelomas versus 1.6-fold for NS/0 control).     

High concentration cultivation of Bifidobacterium bifidum in a submerged membrane bioreactor

In batch cultures, after 25 h, the maximum cell mass of Bifidobacterium bifidum BGN4 was 4.5 g/L, and the maximum cell count was 3.0 x 10(9) cfu/mL at pH 6.0 and 50 g/L sucrose. To increase the viable counts of bifidobacteria, cell retentive culture was applied using a submerged membrane bioreactor with suction and gas sparging. The maximum mass, count, and productivity of the cells after 36 h were 12.0 g/L, 2.2 x 10(10) cfu/mL, and 6.1 x 10(8) cfu/mL x h, respectively, at the feeding (dilution) rate of 120 mL/h (0.06 h-1) in the feeding medium. The accumulated levels of organic acids and ammonium ions at the end of the cultivation were 1.5 and 1.0 g/L, respectively. The viable counts and volumetric productivity of the cells after the cell retentive culture were 7.3- and 5.1-fold higher, respectively, than the values obtained during batch culture. These high viable counts and volumetric productivities were obtained by maintaining lower concentrations of organic acids and ammonium ions so that the growth of B. bifidum BGN4 was not inhibited. The submerged membrane bioreactor produced the highest viable counts of B. bifidum without membrane fouling and cell damage.     

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca²⁺ and Mg²⁺ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl₂ and MgCl₂ concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca²⁺/Mg²⁺ feeding to 117 h with Ca²⁺/Mg²⁺ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 10⁶ and 9.12 × 10⁶ cells ml⁻¹, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.     

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.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

Cell cycle- and growth phase-dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.     

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

One of the key parameters in perfusion culture is the rate of medium replacement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X(v)). The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (Y(Xv/D)) and specific MAb productivity (q(MAb)) remain constant at higher D. To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operated within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems. In the Celligen/vortex-flow filter system, significant reductions in Y(Xv/D) and q(MAb) resulting from the use of gas sparging were observed at D > 1.57 vvd (X(v) > 15 x 10(6) cells/mL). Through glucose supplementation, we have shown that the decrease in Y(Xv/D) encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (q(glc)) and lactate production rates (q(lac)), while no variations were observed in glutamine-consumption rates. As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions. Although a reduction in q(MAb) was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from shear stress-induced increases in q(lac), q(glc), and Y(lac/glc). Deleterious effects of sparging on Y(Xv/D) and q(MAb) encountered in the Celligen/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 x 10(6) viable cells/mL. A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed.     

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.     

Strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies

Summary Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1×10⁶ cells/ml. Concentrations of monoclonal antibody (MAb) reached 140 mg/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during active exponential growth and not during the stationary phase.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation

High-cell-density fed-batch cultures of Pseudomonas putida were carried out for the production of medium-chain-length polyhydroxyalkanoates (PHAs) using oleic acid as a carbon source. By employing an optimal feeding strategy without the limitation of any nutrient, a high cell concentration of 173 g/L was achieved, but the PHA concentration and PHA content were only 32.3 g/L and 18.7 wt%, respectively. To increase the PHA concentration and content, phosphorus limitation was applied during fed-bath culture by reducing the initial KH(2)PO(4) concentration. When the initial KH(2)PO(4) concentration was reduced to 4 g/L, cell concentration, PHA concentration, and PHA content obtained in 38 h were 141 g/L, 72. 6 g/L, and 51.4 wt%, respectively, resulting in a high productivity of 1.91 g PHA/L per hour.     

High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor

A simple feeding strategy was developed and successfully employed for nutritional control in a 2-L fed-batch culture of hybridoma cells. A previously developed stoichiometric model for animal cell growth was used to design a supplemental medium for feeding. Undialyzed fetal bovine serum and trace metals (Fe(2+), SeO(3) (2-), Li(+), Zn(2+), and Cu(2+)) were fed to the cells periodically in addition to the automatic feeding of other nutrients in the supplemental medium. In this study, the maximum viable cell density was increased from 6.3 x 10(6) to 1.7 x 10(7) cells/mL, and the culture span was extended from 340 to 550 hours. The final monoclonal antibody titer achieved was 2400 mg/L. The specific production rates for ammonia and lactate were further reduced from 0.0045 and 0.0048 in our previous fed-batch experiments to 0.0028 and 0.0036 mmol/10(9) cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional batch culture.     

Avoiding rapid growth at high cell densities: A potentially important optimisation criterion for hybridoma cultures

Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002–0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4×10⁹ cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.     

Aggregation effects due to aluminum adsorption to cell walls of the unicellular green alga Scenedesmus obtusiusculus

Cells of the unicellular green alga Scenedesmus obtusiusculus Chod. were cultivated for 2–24 h in nutrient media with low (60 ?mol/L) or high (1000 ?mol/L) phosphorus (P) concentration, and in the presence or absence of 222 ?mol/L aluminum chloride (Al). Cell aggregation was studied by using light microscope, sedimentation and centrifugation. After 2 h, Al was adsorbed to the cell surface and cell aggregates were formed by the attraction of the cells to each other. Aluminum is bound by the negative charges of the cell walls, and studies at different pH showed that a high proportion of positively charged Al forms promote cell aggregation. This effect was most pronounced in low phosphorus cultures as phosphate reduces the effect of Al on cell aggregation by forming aluminum‐phosphate. Algae cultivated in the absence of Al did not show any cell aggregation tendencies.     

杂交瘤细胞培养中摄氧速率(OUR)的在线检测及其与细胞生长及代谢的关系

在批式及灌流培养条件下研究了杂交瘤细胞在无血清培养基中的生长、代谢情况与氧消耗的关系。应用动力学方法在线进行OUR的检测,同时离线取样检测其他参数。结果发现OUR与谷氨酰胺的消耗、抗体的生成及活细胞密度间有明显的相关关系,进一步的分析还发现在对数生长期,OUR与活细胞密度间具有良好的线性关系,qOUR(0.103±0.028)×10^-12mol/cell/h,可以通过它来进行细胞密度的在线检测。并通过以ΔOUR=0时刻作为灌流调整点进行连续灌流培养的初步实验验证了OUR作为培养过程反馈控制参数的可能性。     

Hybridoma cell growth and monoclonal antibody secretion upon continuous cultivation in bioreactor with a serum- and protein-free nutritive medium

Summary 7F11C7 hybridoma cells, secreting a MAb useful for drug targeting, may be cultivated over more than 2 months in bioreactor upon continuous perfusion of serum-, protein-free nutritive medium. With optimal perfusion rate of 48%/day, in the absence of cell recycling, cell density reaches a mean of 2.85 10⁶ viable cells/ml (+ 7% of dead cells). MAb secretion reaches 78 g/ml x day, giving a production of 2.34 g/month x L, i.e. 13 times that obtained in T flasks.     

AcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是一种具有生理调控活性的四肽因子,对造血干/祖细胞增殖具有抑制作用。本研究采用集落形成实验、甲基偶氮唑盐(MTT)比色法、细胞分裂指数测定等方法,考察了AcSDKP对体外培养的人骨髓间充质干细胞(mesenchymal stem cell,MSC)增殖的影响。结果显示,在AcSDKP浓度为1×10-12mol／L-1×10-9mol／L的培养体系中,人骨髓MSC集落生成率和大小、活力细胞数和分裂指数均降低,最大效应浓度为1×10-11mol／L。以上实验结果表明,在体外培养条件下,一定浓度的AcSDKP对人骨髓MSC 的增殖具有抑制作用。     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Acetic acid production using a fermentor equipped with a hollow fiber filter module

The continuous production of acetic acid by Acetobacter aceti M23 was carried out using a fermentor equipped with a hollow fiber filter module. The culture continued for 830 h with various dilution rates, which were changed stepwisely from low to high. The final cell concentration was 21.9 g dry cell/L and the maximum productivity of acetic acid was 12.7 g/L.h for the exit acetic acid concentration of about 50 g/L. The productivity was higher than any literature's values surveyed so far. The cell concentration was 62.8 times and the productivity was 4.6 times as high as those of the fermentor without the filter module. The productivity increased with the increase of dilution rate up to 0.3 h(-1). It is interesting to note that the viable cell concentration was kept almost constant about 1.1 x 10(9) cells/ml in spite of the increase of dilution rate. Use of oxygen-rich air was indispensable to establish the high productivity of acetic acid.