Perfusion bioreactors for the production of recombinant proteins in insect cells

Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.     

Mini-scale bioprocessing systems for highly parallel animal cell cultures

Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.     

High density culture of HeLa cells in a CelliGen perfusion system

A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor. Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems. This system successfully propagated HeLa cells to over 11 million viable cells per milliliter. Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate. Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells. Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor.     

High density cell culture by membrane-based cell recycle

Enhancement of productivity of a bioprocess necessitates continuous operation of bioreactors with high biomass concentrations than are possible in conventional batch, fedbatch or continuous modes of culture. Membrane-based cell recycle has been effectively used to maintain high cell concentrations in bioreactors. This review compares membranebased cell recycle operation with other such high density cell culture systems as immobilized cell reactors and reactors with cell recycle by centrifugation or gravity sedimentation. A theoretical of production of primary and secondary metabolites in membrane-based recycle systems is presented. Operation of this type of system is discussed with examples from aerobic and anaerobic fermentations.     

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent‐pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month‐long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed‐batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913–922, 2017     

Evaluation and applications of optical cell density probes in mammalian cell bioreactors

On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. (c) 1995 John Wiley & Sons, Inc.     

TES and HEPES buffers in mammalian cell cultures and viral studies: Problem of carbon dioxide requirement

In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO₃-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO₂ to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO₂ is required by cells as an essential nutrient for growth, and that the desired level of CO₂ in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO₂ tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO₂.     

A comparison of simple growth vessels and a specially designed bioreactor for the cultivation of hybridoma cells

Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture conditions. The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large differences in vessel and agitator geometry and aeration mode. Furthermore, some data indicate that the hydrodynamic stress level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the reactors.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.     

Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures

Summary A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10⁹ cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O₂ and CO₂ were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10-to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4-and 40-liter versions of this reactor have been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10×10⁶ cells/ml with a total yield of 8.7×10¹¹ cells from 360 liters of medium.     

Design of well and groove microchannel bioreactors for cell culture

Microfluidic bioreactors have been shown valuable for various cellular applications. The use of micro-wells/grooves bioreactors, in which micro-topographical features are used to protect sensitive cells from the detrimental effects of fluidic shear stress, is a promising approach to culture sensitive cells in these perfusion microsystems. However, such devices exhibit substantially different fluid dynamics and mass transport characteristics compared to conventional planar microchannel reactors. In order to properly design and optimize these systems, fluid and mass transport issues playing a key role in microscale bioreactors should be adequately addressed. The present work is a parametric study of micro-groove/micro-well microchannel bioreactors. Operation conditions and design parameters were theoretically examined via a numerical model. The complex flow pattern obtained at grooves of various depths was studied and the shear protection factor compared to planar microchannels was evaluated. 3D flow simulations were preformed in order to examine the shear protection factor in micro-wells, which were found to have similar attributes as the grooves. The oxygen mass transport problem, which is coupled to the fluid mechanics problem, was solved for various groove geometries and for several cell types, assuming a defined shear stress limitation. It is shown that by optimizing the groove depth, the groove bioreactor may be used to effectively maximize the number of cells cultured within it or to minimize the oxygen gradient existing in such devices. Moreover, for sensitive cells having a high oxygen demand (e.g., hepatocytes) or low endurance to shear (e.g., human embryonic stem cells), results show that the use of grooves is an enabling technology, since under the same physical conditions the cells cannot be cultured for long periods of time in a planar microchannel. In addition to the theoretical model findings, the culture of human foreskin fibroblasts in groove (30 microm depth) and well bioreactors (35 microm depth) was experimentally examined at various flow rates of medium perfusion and compared to cell culture in regular flat microchannels. It was shown that the wells and the grooves enable a one order of magnitude increase in the maximum perfusion rate compared to planar microchannels. Altogether, the study demonstrates that the proper design and use of microgroove/well bioreactors may be highly beneficial for cell culture assays.     

Evaluation of criteria for bioreactor comparison and operation standardization for mammalian cell culture

Development of bioprocesses with mammalian cell culture deals with different bioreactor types and scales. The bioreactors might be intended for generation of cell inoculum and production, research, process development, validation, or transfer purposes. During these activities, not only the difficulty of up and downscaling might lead to failure of consistency in cell growth, but also the use of different bioreactor geometries and operation conditions. In such cases, criteria for bioreactor design and process transfer should be carefully evaluated in order to select appropriate cultivation parameters. In this work, power input, mixing time, impeller tip speed, and Reynolds number have been compared systematically for the cultivation of the human cell line AGE1.HN within three partner laboratories using five different bioreactor systems. Proper operation ranges for the bioreactors were identified using the maximal cell‐specific growth rate (μ_max) as indicator. Common optimum values for process transfer criteria were found in these geometrically different bioreactors, in which deviations of μ_max between cultivation systems can be importantly reduced. The data obtained in this work are used for process standardization and comparability of results obtained in different bioreactor systems, i.e. to guarantee lab‐to‐lab consistency for systems biology approaches using mammalian cells.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

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.     

Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line.     

Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments

A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex-centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N-glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred-tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N-glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 10⁶ and 120 × 10⁶ cells/mL and a low cell-specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry.     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.