Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: part I. Cell density, viability, and cell-cycle distribution

For the development of optimal perfusion processes the effect of the feed and bleed rate on cell growth in a perfusion bioreactor was studied. The viable-cell density, viability, growth, death, and lysis rate and cell-cycle distribution of a hybridoma cell line producing an IgG1 were studied over a range of specific feed and bleed rates. It was found that the feed and bleed rates applied in the different cultures could be divided into two regions based on the viable-cell density and cell-cycle distribution. The cultures in the first region, low feed rates (0.5 and 1.0 d(-1)) combined with low bleed rates (0.05 and 0.10 d(-1)), were nutrient-limited, as an increase in the feed rate resulted in an increase in the viable-cell density. The cultures in the second region, high feed and bleed rates, were nonnutrient-limited. In this region the viable-cell density decreased more or less linearly with an increase in the bleed rate and was independent of the feed rate. This suggests that the cells were limited by a cell-related factor. Comparison of Trypan-blue dye-exclusion measurements and lactate-dehydrogenase activity measurements revealed that cell lysis was not negligible in this bioreactor set-up. Therefore, lactate-dehydrogenase activity measurements were essential to measure the death rate accurately. The specific growth rate was nearly constant for all tested conditions. The viability increased with an increase of the bleed rate and was independent of the feed rate. Furthermore, the specific productivity of monoclonal antibody was constant under all tested conditions. For the optimal design of a perfusion process it should first be established whether viability is an important parameter. If not, a bleed rate as low as possible should be chosen. If low viabilities are to be avoided, the bleed rate chosen should be higher, with the value depending on the desired viability. Next, the feed rate should be set at such a rate that the cells are just in the nonnutrient-limited region.     

Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: metabolic analysis

For the development of optimal perfusion processes, insight into the effect of feed and bleed rate on cell growth, productivity, and metabolism is essential. In the here presented study the effect of the feed and bleed rate on cell metabolism was investigated using metabolic flux analysis. Under all tested feed and bleed rates the biomass concentration as calculated from the nitrogen balance (biomass-nitrogen) increased linearly with an increase in feed rate, as would be expected. However, depending on the size of the feed and bleed rate, this increase was attained in two different ways. At low feed and bleed rates (Region I) the increase was obtained through an increase in viable-cell concentration, while the cellular-nitrogen content remained constant. At high feed and bleed rates (Region II) the increase was attained through an increase in cellular-nitrogen content, while the cell concentration remained constant. Per gram biomass-nitrogen, the specific consumption and production rates of the majority of the nutrients and products were identical in both regions, as were most of the fluxes. The major difference between the two regions was an increased flux from pyruvate to lactate and a decreased flux of pyruvate toward citrate in region II. The decreased in-flux at the level of citrate can either be balanced by a decreased out-flux toward lipid biosynthesis leading to a lower fraction of lipids in the cell, by a decreased out-flux toward the citric acid cycle resulting in a decreased energy generation, or by a combination of these. Finally, the specific productivity increases less than the nitrogen content per cell in region II, which implies that for obtaining maximum production rates it is important to increase the cell density and not only the biomass density.     

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Process design and development of a mammalian cell perfusion culture in shake-tube and benchtop bioreactors

The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCD_max), minimum cell-specific perfusion rate (CSPR_min), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell⁻¹·day⁻¹) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 10⁶ cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Long-term perfusion culture of hybridoma: a "grow or die" cell cycle system

In order to elucidate the hybridoma life cycle and the limiting factors in perfusion systems, we performed cultures in a stirred tank bioreactor, coupled to an external tangential flow filtration unit. Cell density and antibody production in perfusion were consistent with previous studies. The average life span of the cells (2.1-2.2 days), antibody, productivity per cell produced (30-38 mg/10(9) cells) and cell size diameter evolution appeared similar to values observed in batch cultures. These observations highly suggest a similar "grow or die" life cycle. Cell and antibody production, strictly related to the medium perfusion rate, seem to be under the control of the nutrient availability. A hypothesis to explain such a life cycle of hybridoma cells in perfusion systems and a model for viable and dead cell density is proposed.     

Stable hybridoma cultivation in a pilot-scale acoustic perfusion system: long-term process performance and effect of recirculation rate

Perfusion systems have the possibility to be operated continuously for several months. It is important that the performance of the cell retention device does not limit the operation time of a perfusion process used in the production of active pharmaceutical ingredients. Therefore, the aim of this study was to investigate the reliability and long-term stability of an acoustic perfusion process using the 200 L/d BioSep. As the BioSep is an external device, it is possible that dependent on the recirculation rate nutrient gradients occur in the external loop, which could affect the cell metabolism. Therefore, the effect of possible nutrient gradients on cell metabolism, viability and productivity was studied by varying the recirculation rate. In this study, it is shown that a perfusion process using a pilot-scale acoustic cell-retention device (200 L/d) is reliable and simple to operate, resulting in a stable 75-day cultivation of a hybridoma cell line producing a monoclonal antibody. The recirculation rate had a significant effect on the oxygen concentration in the external loop, with oxygen being depleted within the cell-retention device at recirculation rates below 6 m3/m(reactor)3.d (=600 L/d). The oxygen depletion at low circulation rates correlated with a slightly increased lactate production rate. For all other parameters no effect of the recirculation rate was observed, including cell death measured through the release of lactate dehydrogenase and specific productivity. A maximum specific productivity of 12 pg/cell.d was reached.     

High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10⁷ cells/ml, when the specific perfusion rate was set to 2.3 vol day^-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel acid proteinase relased by hybridoma cells

An acid proteinase has been detected in culture supernate of the 9.2.27 murine hybridoma. This enzyme extensively degrades albumin and transferrin during short incubations at pH 3 and below. Limited proteolysis of the 9.2.27 IgG_2a appears to occur in the culture supernate. Proteolysis is enhanced at low pH in the presence of urea or 1 M acetic acid. The proteinase activity accumulates in continuous perfusion, total cell recycle cultures, beginning during exponential growth of the hybridoma. It is destroyed by boiling and blocked by pepstatin, but not by inhibitors of cysteine or serine proteinases or by EDTA. The low pH optimum may distinguish this enzyme from the known rat and mouse aspartic acid proteinases including cathepsin D and cathepsin E.A preliminary report of these findings was presented at the 196th National Meeting, American Chemical Society, Los Angeles, September 25–30, 1988; paper #140, Division of Microbial and Biochemical Technology.     

Transferrin recycling perfusion culture of hybridoma cells

A perfusion culture of hybridoma cells in serum-free medium recycling transferrin was carried out, which greatly reduced the level of transferrin that was needed. The culture was maintained even without supplying transferrin for nine days. IgG concentration reached 1.1 mg ml⁻¹ in a month of recycling and its ratio to the total protein was 45.8%. The affinity of the antibody did not decrease and no degradation was observed after long recycling period. The cell density under recycling condition was 2≈3 times higher than that without recycling. It was indicated that there was autocrine growth promoting activity in the culture supernatant.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.     

High-density continuous cultures of hybridoma cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) for animal cell culture was developed and its use in continuous highdensity cultures of hybridoma cells was investigated. In the DFPS, based on cell immobilization in a cylindrical depth filter matrix, cells were easily immobilized and cultivated by simple medium recirculation. The cell density in the 20-mum pore size filter matrix reached up to 3 x 10(7) cells/mLin less than 10 days. This resulted in a high monoclonal antibody productivity of 744 mg/L/day, which was 25-35 times higher than that of continuous-suspension cultures using the same cell line. The 20-mum pore filter retained more cells than the 30-mum filterin a shorter period. The DFPS provides advantages of low-cost set-up, easy operation, and scale-up in the cultures of anchorage-independent cells. It also has a high potential for anchorage-dependent cell cultures because of its unusually high surface-to-volume ratio of 450-600 cm(2)/cm(3). (c) 1994 John Wiley & Sons, Inc.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium

The steady-state metabolic parameters for a murine hybridoma cell line have been determined in continuous suspension culture over a wide range of dilution rates. Long-term adaption occurred over seven months in culture and resulted in lower glucose consumption rates, reduced lactate production, higher cell viability, and, consequently, growth rates more nearly matching the dilution rate. Antibody production rates decreased over the first two months and then remained stable for at least 75 days. The antibody production rate was not found to be growth associated. Steadystate amino acid uptake rates are presented for a wide range of growth rates.     

Determinants and rate laws of growth and death of hybridoma cells in continuous culture

Experimental data from six hybridoma cell lines grown under diverse experimental conditions in both normal continuous and perfusion cultures are analyzed with respect to the significance of nutrients and products in determining the growth and death rates of cells and with respect to their mathematical modeling. It is shown that neither nutrients (glucose and glutamine) nor the common products lactic acid, ammonia, and monoclonal antibody can be generally assumed to be the clear-limiting or inhibiting factors for most of the cultures. Correspondingly, none of the unstructured models existing in the literature can be generally applied to describe the experimental data obtained over a relatively wide range of cultivation conditions as considered in this work. Surprisingly, for all cultures the specific growth rate (mu) almost linearly correlates with the ratio of the viable cell concentration (NV) to the dilution (perfusion) rate (D). Similarly, the specific death rate (kd) is a function of the ratio of the total cell concentration (Nt) to the dilution (perfusion) rate. These results strongly suggest the formation of not yet identified critical factors or autoinhibitors that determine both the growth and death rates of hybridoma cells. Based on these observations, simple kinetic models are developed for mu and kd which describe the experimental data satisfactorily. Analysis of the experimental data with the kinetic models reveals that under the current cultivation conditions the formation rate of the autoinhibitor(s) or the sensitivity of cell growth and death to the autoinhibitor(s) is mainly affected by the medium composition. Irrespective of the cell lines, cells grown on serum-containing media have almost the same model parameters, which are distinctively different from those of cells grown on serum-free media. Furthermore, in contrast to the prevailing view, kd is shown to positively correlate with mu if the effects of cell concentration and dilution (perfusion) rate are considered. Several important implications of these findings are discussed for the optimization and control of animal cell culture.     

Effects of ammonium ion removal on growth and MAb production of hybridoma cells

Production of monoclonal antibody against hepatitis B surface antigen was carried out by perfusion culture coupled with a selective removal system for ammonium ion. The removal system is composed of three sub-systems namely, cell separation by cross-flow ceramic filter, dialysis by hollow fiber module and ion-exchange by zeolite A-3 packed bed column. The ammonium ion concentration in the culture broth was effectively maintained below the inhibitory level, and the viable cell density reached 2.5×10⁷ cells ml^–1 which was three times that of conventional perfusion cultures. The monoclonal antibody accumulated to a concentration as high as 26.3×10⁵ mIU^–1. This is already almost half of the amount producedin vivo. The numerical investigation of the ammonium ion removal system showed the possibility to improve much more the performance of this perfusion cultivation system.     

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Growth characteristics in fed-batch culture of hybridoma cells with control of glucose and glutamine concentrations

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 x 10(6) cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation. (c) 1994 John Wiley & Sons, Inc.