Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.     

Experience in scale-up of homogeneous perfusion culture for hybridomas

A perfusion system for production of monoclonal antibodies was developed using an externally-mounted, hollow-fibre cartridge. The experimental apparatus was operated for 420 h and demonstrated increased steady-state viable cell concentration with increase in perfusion rate. Antibody titres were up to three times those measured for batch cultures and specific antibody productivity was doubled.The procedure was successfully scaled to a 10 dm³ system which produced antibody under conditions of Good Manufacturing Practice (GMP). A calculation of productivity between the scaled perfusion system and 260 dm³ batch cultures resulted in comparable antibody production, whereas the perfusion allowed a halving in medium utilisation. Reactivity assays conducted on the purified antibody from both batch and perfusion cultures showed no evidence of proteolysis or altered antibody activity in the final perfusion product. This study provides additional support for the use of homogeneous perfusion cultures in production of monoclonal antibodies under GMP conditions.     

High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor

Since it was first introduced in late 1990s Wave bioreactor has been used for protein production by mammalian and insect cell lines. However, using Wave bioreactor to produce human monoclonal antibody by stable Drosophila Schneider 2 (S2) cell transfectants has not been reported before. In this study, S2 cells were co-transfected with an inducible vector expressing human monoclonal antibody heavy and light chains, respectively, specific for hemagglutinin (HA) of H5N1 influenza virus. Stable S2 transfectant clone was selected by limiting dilution assay. Stable S2 transfectant clone that produce the highest amount of human monoclonal antibody was inoculated into two 2-l disposable cellbags, where cell growth and antibody production were compared between batch and perfusion cultures using Wave bioreactor. Here, we report that maximum viable cell density reached 1.06?×?10(7) cells/ml in batch culture; whereas 1.04?×?10(8)?cells/ml was achieved in perfusion culture. The maximum volumetric antibody productivity in batch culture was 52?mg/l/day; while perfusion culture yielded 1,437?mg/l/day. As a result, the total antibody production was 201?mg in batch culture and 8,212?mg in perfusion culture. The antibody produced by both cultures displays full neutralizing activity. Thus, our results provide strong support for using Wave bioreactor in perfusion culture for a large-scale production of human monoclonal antibody by stable S2 cell transfectants.     

Biomass and lipid enhancement in Ankistrodesmus sp. cultured with reused and minimal nutrients media

Microalgae are a promising feedstock for biofuel production. Lipid content in microalgae could be enhanced under nutrient depletion. This work investigated the effect of the nutrient on lipid accumulation in Ankistrodesmus sp. culture. Batch cultures were carried out using fresh BG11 medium, and after the harvest, the medium was reused for the next culture; this method was repeated two times. The maximum lipid productivity of 29.75 mg L^?1 day^?1 was obtained from the culture with the second reuse medium. In continuous cultures, Ankistrodesmus sp. was cultured in both fresh and modified BG11 mediums. The modified BG11 medium was adjusted to resemble the content of the first reuse medium. As a comparison between batch and continuous cultures, it was proven that the productivity in the continuous culture was better than in the batch, where the achievable maximum biomass and lipid were 188.30 and 38.32 mg L^?1 day^?1. The maximum lipid content of 34.22% was obtained from the continuous culture at a dilution rate of 0.08 day^?1, whereas the maximum saturated and unsaturated fatty acid productivities of 79.96 and 104.54 mg L^?1 day^?1 were obtained at a dilution rate of 0.16 day^?1.     

Justification of continuous packed-bed reactor for retroviral vector production from amphotropic ΨCRIP murine producer cell

To indentify a plausible large-scale production system forretroviral vector, three culture systems, i.e., batch culturewith medium exchange, microcarrier culture, and packed-bedreactor culture were compared. In batch cultures with mediumexchange, high cell concentrations were maintained for about amonth, and the harvested retroviral titer remained constant. Inmicrocarrier cultures, although cell growth was rapid, theretroviral titer was unexpectedly low, suggesting that the lowtiter was due either to serious damage to the retroviral vectoror to a reduction in the production rate of retroviral vector,caused by mechanical shear forces. Although the retroviral titer(maximum titer, 1.56 × 10⁶) in the packed-bedreactor was a little bit lower than that obtained in the batchculture with medium exchange (maximum titer, 1.91 ×10⁶), continuous production made it possible to increasethe cumulative titer up to 16-fold of that from the batchculture with medium exchange. Moreover, as the packed-bedreactor system requires less labor and shows excellentvolumetric productivity in comparison to batch cultures withmedium exchanges, it will be an appropriate production systemfor retroviral vector in large quantities.     

Production of immunoglobulin A in different reactor configurations

A murine hybridoma line (Zac3), secreting an IgA monoclonal antibody, was cultivated in different systems: a BALB/c mouse, a T-flask, a stirred-tank bioreactor and a hollow fiber reactor. These systems were characterized in terms of cell metabolism and performances for IgA production. Cultures in T-flask and batch bioreactor were found to be glutamine-limited. Ammonia and lactate were produced in significant amounts. IgA productivity was found to be constant and growth associated. Final IgA concentration was similar in both systems. In fed-batch cultures, supplemented with glutamine and glucose, maximum viable cell concentration was increased by 60% and final IgA concentration by 155%. The hollow fiber reactor was able to produce very large amounts of IgA at very high concentrations, similar to the value found in ascites fluid. The productivity ofZac3 is similar to the values reported for IgG-producing cell lines.     

Perfusion seed cultures improve biopharmaceutical fed‐batch production capacity and product quality

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof‐of‐concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high‐seed fed‐batch production cultures. First, we optimized the perfusion N‐1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N‐1 duration, reaching >40 × 10⁶ vc/mL at the end of the perfusion N‐1 stage. The cultures were subsequently split into high‐seed (10 × 10⁶ vc/mL) fed‐batch production cultures. This strategy significantly shortened the culture duration. The high‐seed fed‐batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low‐seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N‐1 and high‐seed fed‐batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low‐seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:616–625, 2014     

Achieving a high-density oleaginous yeast culture: Comparison of four processing strategies using Metschnikowia pulcherrima

Microbial lipids have the potential to displace terrestrial oils for fuel, value chemical, and food production, curbing the growth in tropical oil plantations and helping to reduce deforestation. However, commercialization remains elusive partly due to the lack of suitably robust organisms and their low lipid productivity. Extremely high cell densities in oleaginous cultures are needed to increase reaction rates, reduce reactor volume, and facilitate downstream processing. In this investigation, the oleaginous yeast Metschnikowia pulcherrima, a known antimicrobial producer, was cultured using four different processing strategies to achieve high cell densities and gain suitable lipid productivity. In batch mode, the yeast demonstrated lipid contents more than 40% (w/w) under high osmotic pressure. In fed-batch mode, however, high-lipid titers were prevented through inhibition above 70.0 g L⁻¹ yeast biomass. Highly promising were a semi-continuous and continuous mode with cell recycle where cell densities of up to 122.6 g L⁻¹ and maximum lipid production rates of 0.37 g L⁻¹ h⁻¹ (daily average), a nearly two-fold increase from the batch, were achieved. The findings demonstrate the importance of considering multiple fermentation modes to achieve high-density oleaginous yeast cultures generally and indicate the limitations of processing these organisms under the extreme conditions necessary for economic lipid production.     

Optimization of monoclonal antibody production: combined effects of potassium acetate and perfusion in a stirred tank bioreactor

To increase the yield of monoclonal antibody in a hybridoma culture, it is important to optimize the combination of several factors including cell density, antibody productivity per cell, and the duration of the culture. Potassium acetate enhances the production of antibodies by cells but sometimes depresses cell density. The production of anti-(human B-type red blood cell surface antigen) antibody by Cp9B hybridoma was studied. In batch cultures, potassium acetate inhibited Cp9B cells growth and decreased the maximal cell density but the productivity of antibody per cell was increased. The balance of the two effects resulted in a slight decline of antibody production. In a stirred tank bioreactor, the inhibitory effect of potassium acetate on cell density was overcome by applying the perfusion technique with the attachment of a cell-recycling apparatus to the bioreactor. In such a reactor, potassium acetate at 1 g l^-1 did not cause a decrease in the cell density, and the antibody concentration in the culture supernatant was increased from 28 μg ml^-1 to 38 μg ml^-1. Potassium acetate also suppressed the consumption of glucose and the accumulation of lactate in batch cultures, but the glucose and lactate levels were kept stable by applying the perfusion technique in the stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells

A prolonged period of high productivity at high cell density is desirable for industrial production of biopharmaceuticals. Previous efforts have shown that cessation of cell proliferation in low cell density culture results in increased productivity. We report here further results on multigenic manipulation of cell cycle and apoptosis to enhance productivity at high cell density. The NS0 6A1/4-9F myeloma cell line, which constitutively expresses a chimeric IgG4 antibody and inducibly expresses the p21(CIP1) cyclin-dependent kinase inhibitor has been further engineered to constitutively overexpress the Y28 mutant Bcl-2 anti-apoptotic protein. The effects of overexpression of p21(CIP1) and Bcl-2 on cell proliferation, cell viability, and antibody production has been investigated in batch and continuous perfusion cultures. In both cultures the p21(CIP1) protein arrested cell proliferation, confirming the previous results in low-density culture of 4-fold increase in antibody production, whereas mutant Bcl-2 expression has not resulted in any significant improvement in cell viability of arrested cells. This study demonstrates that it is possible to enhance the productivity of relatively high-density continuous mammalian cell cultures by arresting the cell cycle in G1 phase.     

Predominant growth of α-amylase regulation mutant in continuous culture of Bacillus caldolyticus

The production of α-amylase in batch and continuous cultures of the strain SP of Bacillus caldolyticus was studied using a maltose-casitone medium. The three quantitative parameters of α-amylase production (maximum values of the specific production rate, volumetric productivity and concentration of α-amylase) increased in continuous culture by 2.5, 4.6 and 3.8 times respectively in comparison with a batch culture. It was found that a mutant strain M1 of α-amylase production was predominant in every run of the continuous cultures. The strain M1 differs from the strain SP in that it can produce α-amylase constitutively in a batch culture while the cells of strain SP require maltose for α-amylase production. On the contrary, α-amylase production by the strain M1 was repressed partly by maltose. Moreover, glucose repression on α-amylase production was not observed for the M1 strain while it was remarkable for the strain SP. The above-mentioned properties of the mutant M1 concerning the regulation of α-amylase production are not only advantageous for industrial use but also interesting from the viewpoint of basic microbiology.     

Cultivation of Thermoanaerobium brockii in continuous culture with complete cell recycling

The growth behaviour of the thermophilic anaerobic bacterium Thermoanaerobium brockii for the production of its intracellular secondary alcohol dehydrogenase (sADH) has been studied in batch cultures as well as in continuous cultivation with complete cell recycling. In batch culture the maximum specific growth rate, μ_MAX, was 0·5 h⁻¹, resulting in a cell density of 1·2 g l⁻¹ and an sADH activity of 1·3 units ml⁻¹. Higher glucose concentrations resulted in a decrease in ep cf7 max rs, enzyme productivity as well as biomass yield although an increase in total biomass was achieved. To improve cell density and productivity, continuous culture with complete cell recycling was used, resulting in an increase in cell density by 5 times and in productivity of the sADH by 3 times in comparison to those obtained in batch culture.     

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology

Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5×10⁷ cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.     

Improved production of viable cells of the salt-tolerant yeast Zygosaccharomyces rouxii by continuous culture

Summary A continuous culture system of the salt-tolerant yeast Zygosaccharomyces rouxii (soy yeast) was investigated in order to obtain high production efficiency of viable cells. The optimum pH and C/N ratio of the feed medium for cell production were about 5.0 and 16–20, respectively. About a fivefold increase in viable cell number and cell productivity (viable cell number per litre per hour) were obtained in glucose-limited culture at a dilution rate (D) of 0.06 h^–1 as compared with batch culture. However, the fermentative activity of the cells from glucose-limited culture was significantly lower than those from batch and dissolved-oxygen (DO)-limited cultures, and the former cells showed lower specific activity of glycolytic enzymes. On the other hand, at the boundary conditions between glucose and DO limitation almost the same cell productivity and higher fermentative activity of the cell were obtained as compared with glucose-limited conditions. The cultivation continued for about 60 days without any problems even if the D was altered. It was found that the continuous cultivation method was suitable for industrial production of viable cells of soy yeasts. Offprint requests to: T. Hamada     

Mathematical modeling and analysis of monoclonal antibody production by hybridoma cells

An attempt has been made to mathematically describe and analyze monoclonal antibody (MAB) productivity of hybridoma cells, with particular emphasis on continuous cultures under unsteady-state conditions. A simple and unstructured general kinetic model that takes account of productivity loss during long-term cultivation, cell proliferation, and the effects of nutrients and toxic products is proposed. The model is verified with data of continuous culture from five different cell lines under a wide range of experimental conditions. Analysis of these results showed that for a reliable assessment of effects of different factors and for comparison of kinetic data on MAB production it is important to consider possible loss of MAB productivity, the time dependence of which can be modeled by an exponential function plus a constant term. Variations of nutrient concentration, particularly that of glucose, glutamine, and serum, can significantly alter MAB production under certain conditions. These effects can be described in terms of saturation kinetic and/or noncompetitive inhibition kinetics. (c) 1996 John Wiley & Sons, Inc.     

Production of Streptokinase in Continuous Culture

A method for continuous cultivation of a β-hemolytic streptococcus, strain H 64, is described. The production of cells and streptokinase at various dilution rates, pH, and temperature were studied in a complex medium supplied with excess glucose. At pH 7.0, productivity of cells and streptokinase, as well as the yield constant with respect to glucose, all increased with increasing dilution rate in the range of 0.1 to 0.5 hr^-1. The production of streptokinase was found to be a function of both growth rate and cell concentration. Although higher concentrations of streptokinase were obtained in experiments with batch cultures, the production of streptokinase in continuous cultures was found to be 2.3 times higher. The possible industrial application of a continuous production method is considered.     

Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles

Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 10⁸ cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.     

Monoclonal antibody production in dialyzed continuous suspension culture

Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d(-1). Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.