Reduction of medium consumption in perfusion mammalian cell cultures using a perfusion rate equivalent concentrated nutrient feed

Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 10⁶ cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L⁻¹ day⁻¹ during a short stable phase, and versus 0.46 g L⁻¹ day⁻¹ in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing set-up to enable better control on nutrient addition.     

Recombinant antibody production by perfusion cultures of rCHO cells in a depth filter perfusion system

Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.     

Insights into adenoviral vector production kinetics in acoustic filter-based perfusion cultures

One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.     

Kinetics of prourokinase production by human kidney cells in culture

The kinetics of prourokinase production by human kidney cell line TCL were investigated using microcarriers, roller bottles and a ceramic system. The type of microcarriers used for cell cultivation affects not only growth kinetics but also the duration of post-confluent production period. Different clones of cells derived from the same line can also exhibit different growth kinetics on different types of microcarriers. The effect of serum concentration on the prourokinase production was examined. Using a low serum concentration of 1 % the production of pro-urokinase on microcarriers, roller bottles and a ceramic system was compared. In all three systems the production was sustained over an extended period reaching beyond confluence.     

Continuous medium perfusion leads to long-term cell viability and oxygen production in high-density photobioreactors

Summary A light-emitting diode-based photobioreactor (LED-based PBR) operated in a continuous perfusion mode with a perfusion rate of 3 to 6 reactor volumes a day supports high-density algal cultures, of cell concentrations up to 4·10⁹ cells/mL, or 25 g/L. The oxygen production rate at its peak was 13 to 15 mmol/(L·h). Continuous medium perfusion allowed for long-term stable oxygen production, while oxygen production in batch mode ceased when stationary phase was reached.     

Metabolic flux analysis of HEK-293 cells in perfusion cultures for the production of adenoviral vectors

To meet increasing needs of adenovirus vectors for gene therapy programs, development of efficient and reproducible production processes is required. Perfusion cultures were employed to allow infection at greater cell concentrations. In an effort to define culture conditions resulting in enhanced productivities, experiments performed at different feed rates and infected at various cell densities were compared using metabolic flux analysis. The highest specific product yields were achieved in experiments performed at high perfusion rates and/or low cell concentrations. The intracellular flux analysis revealed that these experiments exhibited greater glycolytic fluxes, slightly higher TCA fluxes, and greater ATP production rates at the time of infection. In contrast, cultures infected at high cell density and/or low medium renewal rates were characterized by a more efficient utilization of glucose at the time of infection, but the specific product yields achieved were lower. The intracellular flux analysis provided a rational basis for the implementation of a feeding strategy that allowed successful infection at a density of 5x10(6)cells/ml.     

Glucose-based optimization of CHO-cell perfusion cultures

Perfusion cultures of CHO cells producing t-PA were performed using acoustic filter cell retention. A robust off-line glucose analysis and predictive control protocol was developed to maintain the process within approximately 0.5 mM of the glucose set point, without the need for a more fallible on-line sensor. Glucose usage (the difference between the inlet and reactor glucose concentrations) provided an easily measured indicator of overall medium utilization for mapping acceptable ranges of operation, including the edge of failure. Earlier onset of perfusion with a ramping glucose set point (1.5 mM/d) resulted in improved growth and consistency during the perfusion culture start-up. At steady state, the t-PA concentration variability increased gradually with increasing glucose usage up to approximately 22 mM, then up to 24 mM the variability increased threefold. Peak t-PA concentrations of over 90 mg/L were obtained by controlling at a glucose usage of approximately 24 mM, but these t-PA levels were not sustainable for more than 3 days. A consistent t-PA concentration of 40 mg/L was obtained at a glucose usage of 21.5 mM.     

Continuous production of tissue plasminogen activator from recombinant CHO cells in a depth filter perfusion system

Summary A depth filter perfusion system (DFPS), equipped with a 40-m polypropylene depth filter for cell immobilization, was used for the continuous production of tissue plasminogen activator (t-PA) from recombinant Chinese hamster ovary cells. Final cell density in the DFPS with oxygen control was 1.8×10⁷ cells/mL of the total working volume and maximum t-PA productivity was 2.63 mg/L/day. Dissolved oxygen concentration in the filter matrix was successfully controlled by air sparging and stable operation was possible for more than 20 days.     

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 10⁶ cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Control of IgG glycosylation in CHO cell perfusion cultures by GReBA mathematical model supported by a novel targeted feed,TAFE

The N-linked glycosylation pattern is an important quality attribute of therapeutic glycoproteins. It has been reported by our group and by others that different carbon sources, such as glucose, mannose and galactose, can differently impact the glycosylation profile of glycoproteins in mammalian cell culture. Acting on the sugar feeding is thus an attractive strategy to tune the glycan pattern. However, in case of feeding of more than one carbon source simultaneously, the cells give priority to the one with the highest uptake rate, which limits the usage of this tuning, e.g. the cells favor consuming glucose in comparison to galactose.We present here a new feeding strategy (named ‘TAFE’ for targeted feeding) for perfusion culture to adjust the concentrations of fed sugars influencing the glycosylation. The strategy consists in setting the sugar feeding such that the cells are forced to consume these substrates at a target cell specific consumption rate decided by the operator and taking into account the cell specific perfusion rate (CSPR). This strategy is applied in perfusion cultures of Chinese hamster ovary (CHO) cells, illustrated by ten different regimes of sugar feeding, including glucose, galactose and mannose. Applying the TAFE strategy, different glycan profiles were obtained using the different feeding regimes. Furthermore, we successfully forced the cells to consume higher proportions of non-glucose sugars, which have lower transport rates than glucose in presence of this latter, in a controlled way.In previous work, a mathematical model named Glycan Residues Balance Analysis (GReBA) was developed to model the glycosylation profile based on the fed carbon sources. The present data were applied to the GReBA to design a feeding regime targeting a given glycosylation profile. The ability of the model to achieve this objective was confirmed by a multi-round of leave-one-out cross-validation (LOOCV), leading to the conclusion that the GReBA model can be used to design the feeding regime of a perfusion cell culture to obtain a desired glycosylation profile.     

Continuous cultures of tomato and citron roots in vitro

Summary Initial trials with tomato-root cultures disclosed the desirability of employing a gently agitated liquid medium containing iron in the chelated form. For the normal cultivars “Ace” and “Tropic”, subcultures were best achieved by utilizing sectors that possessed one or more newly emerged laterals. Continuous cultures of a nonlateral-forming tomato mutant, “Diageotropica”, and of citron were accomplished by subculturing tips of the elongating primary roots. The tomato roots were cultured in White's medium with the Fe₂(SO₄)₃ replaced by 0.03 mM NaFeEDTA. Sustained growth of citron-root tips necessitated the use of a medium containing Murashige and Skoog salts, 7.5% sucrose, 100 mg per I each of citric acid and thiamine HCl, and 5000 mg per li-inositol. The success with citron-root cultures was extendable to all cultivars ofC. medica L., but not to otherCitrus species relatives. Both citron and “Diageotropica” root cultures manifested undiminished elongation through repeated subcultures; but neither produced laterals in response to any cultural treatments. Research was supported in part by National Science Foundation Grant OIP75-10390 and Elvenia J. Slosson Fellowship in Ornamental Horticulture.     

血清浓度及溶氧对培养rCHO细胞生产尿激酶原的影响

在30L搅拌式反应器中用多孔微载体培养分泌尿激酶原的DNA重组中国仓鼠卵巢细胞(rCHO),研究了血清浓度及溶氧对细胞生长和表达的影响.结果表明,在2×105/ml低密度接种条件下,使用低(无)血清培养基会延长细胞生长延滞期并降低比生长速率,而细胞密度大于106/ml时,血清浓度对细胞生长和产物表达的影响没有显著差别.溶氧(DO)维持在20%～45%时,对细胞表达产物和葡萄糖代谢无明显影响,但溶氧降至7%～9%时,细胞表达水平明显降低,葡萄糖代谢转化为乳酸的比例上升.     

Glucosyldiacylglycerol enhances reciprocal activation of prourokinase and plasminogen

Reciprocal activation of prourokinase (pro-u-PA) and plasminogen is an important mechanism in the initiation and propagation of local fibrinolytic activity. We found that glucosyldiacylglycerol (GDG) enhanced the reciprocal activation by 1.5- to 2-fold at 0.7-16 microM, accompanying increased conversions of both zymogens to active two-chain forms. The reciprocal activation system consists of (i) plasminogen activation by pro-u-PA to form plasmin, (ii) pro-u-PA activation by the resulting plasmin to form two-chain u-PA (tcu-PA), and (iii) plasminogen activation by the resulting tcu-PA. Whereas GDG minimally affected steps (ii) and (iii) in isolated systems, it markedly enhanced step (i) in the absence of the conversion of pro-u-PA to tcu-PA. GDG significantly increased the intrinsic fluorescence of pro-u-PA (6.7%), but not that of tcu-PA or plasminogen. The large change in intrinsic fluorescence suggests that GDG selectively affects pro-u-PA to alter its conformation, and this mechanism may account for enhancement of its intrinsic plasminogen activator activity.     

Influence of supplemental endoglucanase or xylanase on volatile fatty acid production from ruminant feed by ruminal in vitro cultures

This study employed two commercial enzyme preparations to examine the effects of endoglucanase, xylanase or their combination on in vitro volatile fatty acid (VFA) production by ruminal microbial populations. Batch ruminal cultures were established with one of various feedstuffs or with a fescue hay-based diet and ruminal fluid from a heifer fed a 40% forage:60% concentrate diet. Addition of xylanase at 135 xylanase units (XU) per ml increased total VFA production from the fescue hay-based diet (44.3 vs. 57.2 mM, p < 0.05) without changing the acetate to propionate (A:P) ratio. Addition of endoglucanase at 2, 3, 4, and 5 carboxymethyl cellulase units (CMCU) per ml increased total VFA production from the fescue hay-based diet on average by 36% (p < 0.05). Addition of 3, 4 and 5 CMCU/ml also decreased (p < 0.05) the A:P ratio. The combined addition of xylanase (135 XU/ml) and endoglucanase (5 CMCU/ml) increased total VFA production from the fescue hay-based diet (40.9 vs. 61.5 mM, p < 0.05) and reduced the A:P ratio (3.4 vs. 1.5, p < 0.05). The effects of endoglucanase and xylanase supplementation on in vitro VFA production varied across the various substrates used. However, endoglucanase supplementation consistently reduced the A:P ratio with all substrates tested. The effects of the enzyme combination were generally greater than either enzyme alone. We conclude that endoglucanase and xylanase activities differ in their ability to affect ruminal VFA production, and endoglucanase but not xylanase, may improve fermentation efficiency by reducing the A:P ratio.     

Influence of supplemental endoglucanase or xylanase on volatile fatty acid production from ruminant feed by ruminal in vitro cultures

Abstract

This study employed two commercial enzyme preparations to examine the effects of endoglucanase, xylanase or their combination on in vitro volatile fatty acid (VFA) production by ruminal microbial populations. Batch ruminal cultures were established with one of various feedstuffs or with a fescue hay-based diet and ruminal fluid from a heifer fed a 40% forage:60% concentrate diet. Addition of xylanase at 135 xylanase units (XU) per ml increased total VFA production from the fescue hay-based diet (44.3 vs. 57.2 mM, p < 0.05) without changing the acetate to propionate (A:P) ratio. Addition of endoglucanase at 2, 3, 4, and 5 carboxymethyl cellulase units (CMCU) per ml increased total VFA production from the fescue hay-based diet on average by 36% (p < 0.05). Addition of 3, 4 and 5 CMCU/ml also decreased (p < 0.05) the A:P ratio. The combined addition of xylanase (135 XU/ml) and endoglucanase (5 CMCU/ml) increased total VFA production from the fescue hay-based diet (40.9 vs. 61.5 mM, p < 0.05) and reduced the A:P ratio (3.4 vs. 1.5, p < 0.05). The effects of endoglucanase and xylanase supplementation on in vitro VFA production varied across the various substrates used. However, endoglucanase supplementation consistently reduced the A:P ratio with all substrates tested. The effects of the enzyme combination were generally greater than either enzyme alone. We conclude that endoglucanase and xylanase activities differ in their ability to affect ruminal VFA production, and endoglucanase but not xylanase, may improve fermentation efficiency by reducing the A:P ratio.     

Continuous hypothermic perfusion of rabbit kidneys.

Addition of cryoprotective agents to whole organs is possible only by vascular perfusion with the cryoprotectant dissolved in a suitable perfusion fluid.Vascular resistance, organ weight gain, release of lactate dehydrogenase (LDH), and post-transplant function was studied during and after hypothermic perfusion at +6 °C of rabbit kidneys with six different perfusion fluids. A mixture of dextran and bovine serum albumin (BSA), BSA alone in various concentrations, and human serum albumin were tested as colloids, and the effect of perfusate osmolality was investigated.The dextran-BSA mixture was found to be superior to 4.5 and 6.0% BSA alone in terms of better perfusion characteristics, better post-transplant function, and lower LDH release. Perfusion characteristics during perfusion with human serum albumin and subsequent graft function were not different from those observed in experiments with dextran-BSA, but the LDH release was lower.Perfusate osmolality was increased by the addition of glucose or mannitol. Perfusion characteristics during perfusion with the hypertonic perfusates were not different from those observed during isotonic perfusion, but post-transplant function seemed to be better after perfusion with the fluid made hypertonic with glucose, whereas addition of mannitol seemed to be deleterious.Thus a perfusion fluid of extracellular electrolyte composition, containing human serum albumin as a colloid and made hypertonic with glucose, can be used as a vehicle for cryoprotectants during their addition to rabbit kidneys.     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of ∼5 × 10⁶ cells mL^-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell^-1 day^-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized (∼40 mg L^-1) at 0.2 nL cell^-1 day^-1. The volumetric protein productivity (∼60 mg L^-1 day^-1 was maximized above 0.3 nL cell^-1 day^-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.