Perfusion culture of hybridoma cells for hyperproduction of IgG(2a) monoclonal antibody in a wave bioreactor-perfusion culture system

A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.     

Application of a cell-once-through perfusion strategy for production of recombinant antibody from rCHO cells in a Centritech Lab II centrifuge system

Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.     

Impact of plasminogen on an in vitro wound healing model based on a perfusion cell culture system

Vascular reorganization in wound healing is a complex process, which involves coagulation, endothelial cell proliferation and migration, basement membrane regeneration, and fibrinolysis. During this healing process, the hemostatic system and the angiogenic system are intimately interconnected. To elucidate the contribution of plasminogen in the process of wound healing, we have established a perfusion cell culture system. Using this novel cell culture system, we found that addition of plasminogen in the perfusion medium allowed the "scratch-wounded" endothelial cells to recover completely, while mini-plasminogen only affected the migration but not the proliferation of the endothelial cells. In the process of recovery with the addition of plasminogen, significant plasmin activity could only be detected when the growth of the endothelial cells have almost reached confluence. This finding indicates that wound healing is triggered and promoted during the absence of the proteolytic activity of plasmin. In addition, we could not detect any matrix metalloproteinase activity in the perfusion culture medium throughout the whole culture period. However, we did found that the circulating medium collected from the perfusion system at the early phase of the healing process has stimulatory activity on the growth of endothelial cells, but the proliferative activity decreased back to the basal level when the cells reached confluence. Thus, by using the perfusion cell culture system, we found that proliferation of endothelial cells is regulated by plasminogen and the wound healing process is controlled by a temporal interaction between the endothelial cells and plasminogen.     

Three-dimensional cell seeding and growth in radial-flow perfusion bioreactor for in vitro tissue reconstruction

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.     

High density VERO cell culture on microcarriers in a cell bioreactor

VERO cells were cultivated in microcarriers (MCs) using a bioreactor with a working capacity of 3.7 l. We studied the effect of MCs concentration in the proportion of MC-bearing cells and on the kinetics of cell growth, as well as the cell growth with a batch, fed-batch and perfusion mode operation. The data show that, in a batch system, with an initial VERO cell inoculum of 3×10⁴ cells/mg of MCs, 65%, 55% and 50% of the MCs were shown to be totally covered by cells after 7 days in cultures with respectively 2, 5 and 10 mg/ml of MCs. It was observed, that higher concentrations of MCs produced higher final yields of VERO cells. With 10 mg/ml of MCs, after 7 days of culture, a final yield of 10¹⁰ cells in the bioreactor, was obtained. The study of cell growth with a batch, fed-batch or perfusion system showed that the medium renewal allowed the continuous cell growth with the obtention of a final yield of 4×10⁹ cells in a culture with 2 mg/ml of MCs. The number of cells that can be easily reached in these culture systems, which can be even further improved, indicates its usefulness for cell propagation and the preparation of cell products such as viral antigens.This work was supported in part by grants from the Fundação de Amparo à Pesquisa do Estado de S. Paulo (FAPESP), Fundação Instituto Butantan, and European Economic Community (EEC). C.A. Pereira is recipient of a Conselho Nacional de Pesquisa (CNPq) fellowship. We thank A.C. Barbosa for technical assistance.     

The multilayer growth of epithelium-like ESK cells in perfused cultures]

The multilayer growth of an epithelium-like ESK [correction of SPEV] cell culture was achieved under condition of culture medium perfusion. The growth of the ESK [correction of SPEV] cells is described under conditions excluding medium movement above the cells in the chamber with a semipermeable membrane separating the cells from the perfused medium. Under these conditions the multilayer (30-40 layers) growth of culture was observed. The data observed are in favour of our suggestion that it is the diffusional limitations in mass exchange, rather than intracellular interaction contacts, that may be responsible for cell growth in culture. A dependence of culture growth on the initial inoculation density, under condition of medium perfusion immediately above the cells, was determined. The dependence of the multilayer saturation density on the perfusion rate and on the complete replacement of the perfused medium is discussed. The existence of a limiting saturation density of perfused culture was shown.     

High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler

A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.     

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.     

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.     

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

Calcium alginate immobilized hybridomas grown using a fluidized-bed perfusion system with a protein-free medium

Hybridoma SPO1 cells were immobilized in calcium alginate beads and were further grown in a fluidized-bed perfusion system with a protein-free medium. The presence of serum in the steps of entrapment was shown to be helpful for the preservation of cell viability. Each step during immobilization was investigated with respect to the extent of cell damage caused. The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads. In a perfusion system for the continuous production of monoclonal antibodies (MAb), the viable cell density reached 2×10⁷ cells per ml of beads with a viability of 40%. Compared with the cells in suspension culture, the immobilized SPO1 cells showed higher viable cell based specific rates of substrate uptake (glucose and glutamine) and of MAb production. A significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.     

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.     

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.     

A modified matrix perfusion-microcarrier bead cell culture system. I. Adaptation of the matrix perfusion system for growth of human foreskin fibroblasts

Two strains of human foreskin fibroblast cells were incapable of sustained growth in a matrix perfusion culture system, possibly because of their inability to attach to the fiber surfaces. Addition of microcarrier beads to the extracapillary space allowed attaining high cell densities in excess of 10(7) cells per culture unit. Microcarrier beads were tested in hollow fiber culture devices containing membranes of 10(4) or 10(5) D nominal porosities. Best results were obtained when initial cell densities of at least (2-3) x 10(6) cells were used in units with 10(5) D pore size membranes and DEAE-Sephadex or polyacryl-amide microcarrier beads in the extracapillary space. This extension of the matrix perfusion system should be useful for growing other anchorage dependent cells while retaining the advantages of perfusion culture.     

Monoclonal antibody productivity and the metabolic pattern of perfusion cultures under varying oxygen tensions

The metabolic pattern and cell culture kinetics of high-cell-density perfusion cultures were compared under two different oxygen transfer conditions: oxygen limiting and not limiting. When oxygen was a limiting factor during perfusion culture, both specific glucose uptake and lactate production rates increased, compared to non-oxygen-limited condition, by about 60% and 30%, respectively. The specific glutamine uptake rate under oxygen-limited conditions was almost 4.0 times higher than that under non-oxygen-limited conditions. The activity of lactate dehydrogenase (LDH) released into the medium by the dead cells can be used as an indicator for the metabolic and physiological conditions related to oxygen limitation. There was a 3.2 times higher specific rate of LDH activity released by dead cells in oxygen-limited cultures than those in non-oxygen-limited cultures. The specific production rate of monoclonal antibody was not significantly affected by the oxygen transfer conditions during the rapid cell growth period, but it rapidly increased toward the end of perfusion cultures. The higher perfusion rate may have limited further cell growth during high-cell-density perfusion culture, because cell damage was caused by the hydrodynamic shear within a hollow fiber microfiltration cartridge installed to withdraw the spent medium and the waste metabolites. (c) 1993 John Wiley & Sons, Inc.     

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.     

杂交瘤细胞培养中摄氧速率(OUR)的在线检测及其与细胞生长及代谢的关系

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

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

高渗胁迫和灌流培养策略提高HEK 293细胞生产重组腺病毒载体产量

由于各种疾病在全球范围内的肆虐,国际市场对重组腺病毒载体(adenoviral vector,Adv)疫苗的需求量急剧增加,而工艺研究是解决这一问题的有效手段之一。在细胞接毒前施加高渗胁迫可以提高分批培养模式下的Adv产量,新兴的灌流培养也可以显著提高Adv的产量。将高渗胁迫工艺与灌流培养相结合,有望进一步提升高细胞密度生产过程中的Adv产量。本研究利用摇瓶结合拟灌流培养作为生物反应器灌流培养的缩小模型,使用渗透压为300–405 mOsm的培养基研究了高渗胁迫对细胞生长和Adv生产的影响。结果显示,在细胞生长阶段使用370 mOsm的高渗透压培养基,在病毒生产阶段使用300 mOsm的等渗透压培养基的灌流培养工艺有效地提高了Adv的产量。进一步研究发现这可能归因于病毒复制后期HSP70蛋白的表达量增加。将这种工艺放大至生物反应器中,Adv的产量达到3.2×10¹⁰ IFU/mL,是传统灌流培养工艺的3倍。本研究首次将高渗胁迫工艺与灌流培养相结合的策略应用于HEK 293细胞生产Adv,同时揭示了高渗胁迫工艺增产Adv的可能原因,为HEK 293细胞生产其他类型Adv的工艺优化提供了借鉴。