Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications.

This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (>10(8) cells ml(-1)) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice.

Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks.

Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.

Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality.

During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed by a complementary set of studies (e.g. full physico-chemical characterization, assessment of in vitro and in vivo bioactivity, comparative pharmacokinetics and pharmacodynamics studies in relevant species, etc.) would be required. Finally, this illustrates the need to fix the production process early during the development of a new drug product in order to minimize process conversion efforts and to shorten product development time lines.

Use of glucose consumption rate (GCR) as a tool to monitor and control animal cell production processes in packed-bed bioreactors.

For animal cell cultures growing in packed-bed bioreactors where cell number cannot be determined directly, there is a clear need to use indirect methods that are not based on cell counts in order to monitor and control the process. One option is to use the glucose consumption rate (GCR) of the culture as an indirect measure to monitor the process in bioreactors. This study was done on a packed-bed bioreactor process using recombinant CHO cells cultured on Fibra-Cel disk carriers in perfusion mode at high cell densities. A key step in the process is the switch of the process from the cell growth phase to the production phase triggered by a reduction of the temperature. In this system, we have used a GCR value of 300 g of glucose per kilogram of disks per day as a criterion for the switch. This paper will present results obtained in routine operations for the monitoring and control of an industrial process at pilot-scale. The process operated with this GCR-based strategy yielded consistent, reproducible process performance across numerous bioreactor runs performed on multiple production sites.

Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells.

In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel((R)) disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (2.5x10(7) cell ml(-1) of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by -25% and -50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a -25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a -30% loss of productivity was obtained when the medium perfusion rate was further reduced to -50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of -25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufacturing operations.

Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells.

This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes.

[Hodgkin's disease manifesting as paraneoplastic limbic encephalitis]. / Encéphalite limbique révélatrice d'une maladie de Hodgkin.

INTRODUCTION: Malignancy is a possible cause of unexplained encephalitis. EXEGESIS: We describe a 59-years-old woman with limbic encephalitis, not explained by other causes, preceding diagnosis of Hodgkin's disease. Successful treatment of Hodgkin's disease was effective against neurological disturbance. CONCLUSION: This case provides evidence that Hodgkin's disease can be uncovered by paraneoplastic limbic encephalitis.

Monitoring of temperature effects on animal cell metabolism in a packed bed process.

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the data were used for process development. At 37 degrees C cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate production rates increased continually. Reduction of the temperature to 33.5 degrees C resulted in a lowering of these metabolic rates while having no effect on cell proliferation. Subsequent reduction of the temperature to 32 degrees C resulted in stabilization of the cell concentration at a high density (3.6 x 10(7) cell per mL of packed bed). In addition, the specific production rate of the protein of interest increased by a factor of 6 compared to the value at 37 degrees C. During the stationary phase at 32 degrees C, all other specific metabolic rates could be controlled to low and constant levels.

On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy.

Dielectric spectroscopy was applied to two industrial high cell density culture processes and used to determine on-line the concentration of CHO cells immobilized on macroporous microcarriers in a stirred bioreactor and in a packed-bed of disk carriers. The cell concentration predicted from the spectroscopic data was in excellent agreement with off-line cell counting data for both processes. Deviations between the two counting methods only occurred in the case of a significant decrease of the cell viability, from 93% to 64%, which induced a change of the average cell size in the culture. Results for the packed-bed process were further confirmed by the application of indirect yield models based on the measurement of glucose, lactate, and the protein of interest. Moreover, dielectric spectroscopy was used as a tool to characterize the packed-bed process. It was possible to determine both the maximum cell concentration that could be reached in the culture system, 2.0 x 10(11) cell per kg of disk carrier, and to quantify the increase of specific protein productivity induced by the production phase, from 5.14 x 10(-8) microg x cell(-1) x h(-1) to 4.24 x 10(-7) microg x cell(-1) x h(-1).

The effect of octreotide on wound healing: an in vitro and in vivo experimental study.

OBJECTIVE: To investigate the effect of octreotide on wound healing. DESIGN: Experimental studies in vitro and in rats. SETTING: Teaching hospital, Israel. MATERIAL: Cultured human diploid fetal fibroblasts, and 36 male Wistar rats. INTERVENTIONS: Octreotide was added to cultures of fibroblasts in doses of 2, 10, 30, 60 and 120 ng/ml and fibroblasts were counted after 2, 4, and 6 days. Intestinal anastomoses were made in 36 rats. Rats in the octreotide group (n = 18) were given subcutaneous injections of 0.25 microg/kg twice daily and 6 rats were killed at 3, 7, and 14 days. The control group were given injections of saline. Anastomotic bursting pressures and hydroxyproline content were measured at each of the three times. MAIN OUTCOME MEASURES: Fibroblast counts, anastomotic bursting pressures, and hydroxyproline concentrations. RESULTS: Octreotide did not inhibit fibroblast proliferation in any of the doses at any of the time periods. The anastomotic bursting pressure was slightly higher in the octreotide group at each of the time points, but not significantly so, and there was no difference in hydroxyproline content between the octreotide and control groups. Octreotide did not inhibit wound healing either in vitro or in vivo.

Repair of fascia with polyglycolic acid mesh cultured with fibroblasts--experimental study.

In the present study polyglycolic acid (PGA) mesh was used for repair of fascial defects experimentally made in rats. In the experimental group fibroblasts were cultured on the mesh before implantation and in the control group the mesh alone was used. Rats were sacrificed 7, 14, 28 and 60 days after the implantation of the mesh. Tissues were examined microscopically and for hydroxyproline content. Microscopically good incorporation of the mesh was noted in both the control and experimental groups. However, it seems that in the experimental group earlier dissolution of the mesh occurred. The hydroxyproline content was higher in the experimental group after 7 (statistically not significant) and 14 days (p < 0.05) and in the control group after 28 (p < 0.025) and 60 days (p < 0.05). These results suggest that the use of PGA mesh with cultured fibroblasts might have a beneficial effect on wound healing.

Some myths and messages concerning the batch and continuous culture of animal cells.

lt is often assumed that continuous processes are more difficult and less productive than a suite of batch processes for the production of a particular biomolecule. This paper cites two papers which have appeared in the literature which propound this view and examines in detaü the justification for the support of this contention. After reviewing those features where it is alleged that continuous processes are at a disadvantage, the authors of this paper conclude that the opposite is the case and that for suitable processes the most effective way of generating product is by the use of fully continuous processes. The choice of a particular process dependends on a variety of fixed and variable factors which are unique to the process. These factors are discussed and two decision trees are presented which are designed to facilitate the choice of the appropriate process technology.

Mineralization of marrow-stromal osteoblasts MBA-15 on three-dimensional carriers.

The present study describes a new three-dimensional (3-D) culture system that enables the maintenance and phenotypic expression of bone marrow stromal osteoblasts. This culture substratum is advantageous in that it provides suitable conditions for attachment, growth, and differentiation of cells forming 3-D layers. The MBA-15 cell line was grown in unlimited quantities on 3-D Fibro-Cel carriers. These cells mineralized when exposed to ascorbic acid and beta-glycerophosphate (beta GP). Under these mineralization conditions, mRNA expressions of procollagen alpha 2(I) and [3H]-proline-labeled protein were increased. The expression of mRNA for osteonectin and to a lesser extent, for osteopontin was increased, whereas alkaline phosphatase and biglycan remained unaffected under similar conditions. Exposure of mineralizing cultures to dexamethasone reduced mRNA of procollagen alpha 2 (I) and osteonectin to control level. Scanning electron microscopy revealed that cells were grown along the fabric's fibers and produced collagen fibrils. Under appropriate conditions, extensive mineralization had taken place. The mineralization process involves the formation of calcospherites, and correlates with an increase in calcium content. The Fibro-Cel carriers enable formation of 3-D architecture and mineralized tissue in vitro.

Inhibition of tissue plasminogen activators and urokinase by human saliva.

The fibrinolytic activity found in body fluids is mediated through the generation of plasmin from plasminogen by plasminogen activator. Mixed fresh human saliva was found to have plasminogen activator inhibitory activity that has not yet been characterized. In the present study, mixed fresh human saliva was found to contain an inhibitor for tissue plasminogen activator and for urokinase. The plasminogen activator inhibitory activity was mainly found in the mixed fresh human saliva cellular pellet containing buccal epithelial cells and not in the supernatant salivary fluid. Saliva collected by Stenson's duct cannulation does not have any PA-inhibitor activity, although saliva collected from submandibular glands contains small amounts of such activity. The salivary plasminogen activator inhibitor is unstable, and its activity is lost during storage.

Dynamic changes in cytokine secretion by stromal cells during prolonged maintenance under protein-free conditions.

Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities. Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells. We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%). The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable. Stromal cells of the MBA-2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of non-woven fabric matrix. After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in protein-free medium. Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF) and restrictin-P, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells. In addition, interleukin-6 (IL-6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml. Concomitantly, the production of restrictin-P diminished and reached its lowest levels at the end of 10 months. The results may imply a possible causal relationship between the expression of IL-6 and restrictin-P, since no similarly significant changes were observed in the titers of M-CSF and TGF-beta. This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serum-free conditions.

Modified CelliGen-packed bed bioreactors for hybridoma cell cultures.

This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG1 monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 10(8) cells per cm3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1%) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.

Modulation of tissue plasminogen activator biosynthesis by phosphatidylinositol liposomes in human fetal lung fibroblasts.

Phosphatidylinositol (PI) liposomes at 40 microM increased tissue plasminogen activator (t-PA) biosynthesis by human fetal lung fibroblasts IMR-90 (FLF), after 5 days of incubation by 7.4 +/- 1.4 times of the control level. Other phospholipid liposomes, such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylglycerol (PG), had no effect on t-PA biosynthesis by FLF. The induction of t-PA biosynthesis by PI liposomes was inhibited by specific inhibitors of phosphoinositide pathway: gentamycin and lithium chloride. Thus, gentamycin inhibited the effect of PI liposomes on t-PA biosynthesis by 76% (P less than 0.001), while it had no effect on control FLF. Likewise, lithium chloride inhibited t-PA biosynthesis of both PI-treated and control FLF by greater than 84%. The induction of t-PA biosynthesis by PI liposomes was dependent on RNA transcription and independent of DNA biosynthesis.