Perfusion Reactor Research Articles

Integration of a perfusion reactor and continuous precipitation in an entirely membrane-based process for antibody capture.

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl2 precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab-scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand-alone capture and as an integrated perfusion-capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow-through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host-cell proteins and host-cell DNA by the combination of the precipitation-based capture and the first polishing step.

Design and characterization of a novel perfusion reactor for biopharmaceuticals production

The demand for commercially valuable biopharmaceuticals able to treat different immunopathological diseases has accelerated in the last few decades. In recent years the industry has shifted the preferred mode of operation towards continuous strategies and has favoured the adoption of perfusion approaches for the cell culture step. In this mode successful perfusion process examples have reached up to 10-fold higher cell densities and product titres than fed-batch, while maintaining product quality at reduced costs. Perfusion processes are operated at high cell densities where cells are retained, while product and waste are continuously removed. This leads to different requirements in flow and mixing in comparison to lower cell density operations, potentially influencing the cellular production performance and therefore product quality. As the pharmaceutical industry is highly regulated, ensuring homogeneity in the bulk throughout the process is critical, while oxygen and mixing requirements of high cell density cultures must be continuously met. This paper presents the design and characterization of a novel 250 mL stirred tank reactor (STR) developed to work in perfusion mode. The results presented include the experimental measurement of the power consumption, the mixing analysis of the flow within the bioreactor which informs design consideration and scaling efforts and the biological data of the perfusion stage with an industrially relevant cell line producing an IgG monoclonal antibody. The characterization allows the operating window of the reactor to be established, resulting in increased productivity of intensified cultures.

Experimental measurements and CFD modelling of hydroxyapatite scaffolds in perfusion bioreactors for bone regeneration.

In the field of bone tissue engineering, particular interest is devoted to the development of 3D cultures to study bone cell proliferation under conditions similar to in vivo ones, e.g. by artificially producing mechanical stresses promoting a biological response (mechanotransduction). Of particular relevance in this context are the effects generated by the flow shear stress, which governs the nutrients delivery rate to the growing cells and which can be controlled in perfusion reactors. However, the introduction of 3D scaffolds complicates the direct measurement of the generated shear stress on the adhered cells inside the matrix, thus jeopardizing the potential of using multi-dimensional matrices. In this study, an anisotropic hydroxyapatite-based set of scaffolds is considered as a 3D biomimetic support for bone cells deposition and growth. Measurements of sample-specific flow resistance are carried out using a perfusion system, accompanied by a visual characterization of the material structure. From the obtained results, a subset of three samples is reproduced using 3D-Computational Fluid Dynamics (CFD) techniques and the models are validated by virtually replicating the flow resistance measurement. Once a good agreement is found, the analysis of flow-induced shear stress on the inner B-HA structure is carried out based on simulation results. Finally, a statistical analysis leads to a simplified expression to correlate the flow resistance with the entity and extensions of wall shear stress inside the scaffold. The study applies CFD to overcome the limitations of experiments, allowing for an advancement in multi-dimensional cell cultures by elucidating the flow conditions in 3D reactors.

Dynamic Optimization for Monoclonal Antibody Production

This paper presents a dynamic optimization numerical case study for Monoclonal Antibody (mAb) production. The fermentation is conducted in a continuous perfusion reactor. We represent the existing model in terms of a general modeling methodology well-suited for simulation and optimization. The model consists of six ordinary differential equations (ODEs) for the non-constant volume and the five components in the reactor. We extend the model with a glucose inhibition term to make the model feasible for optimization case studies. We formulate an optimization problem in terms of an optimal control problem (OCP) and consider four different setups for optimization. Compared to the base case, the optimal operation of the perfusion reactor increases the mAb yield with 44% when samples are taken from the reactor and with 52% without sampling. Additionally, our results show that multiple optimal feeding trajectories exist and that full glucose utilization can be forced without loss of mAb formation.

Dynamic simulation, optimisation and economic analysis of fed-batch vs. perfusion bioreactors for advanced mAb manufacturing

The variation of antigen binding sites on monoclonal antibodies (mAbs) enables manufacturing of the latter for targeted treatment of a variety of ailments, including cancer types and autoimmune diseases. Fed-batch operation of biochemical reactors used to manufacture mAbs grants the ability to manipulate cell dynamics via intermittent feeding of nutrients/substrates. Regulatory authority agendas (e.g. the U.S. Food & Drug Administration) consider the development of continuous technologies (e.g. perfusion reactors) as essential towards improved technoeconomic potential and biopharmaceuticals quality control. This paper presents dynamic simulations for multiple operational scenaria of a fed-batch and a perfusion reactor, in which a hybridoma cell culture is used to secrete mAb glycoforms. A novel dynamic optimisation of the fed-batch as well as perfusion reactors has also been conducted towards maximising the total mAb mass produced. Nonlinear Programming (NLP) formulations with the use of APOPT and IPOPT solvers have been employed to derive the manipulation, state and controlled variable trajectories, to achieve the mAb production maximisation objective. The detailed technoeconomic analysis clearly highlights strong benefits for the fed-batch bioreactor (especially over a plant-life time horizon), but also showcases a definite cost-related promise of perfusion bioreactor technologies, given the higher mAb yield.

Development of a Perfusion Reactor for Intervertebral Disk Regeneration.

A tissue-engineered biological disk replacement has been proposed as a promising approach for the treatment of degenerative disk disease. A perfusion bioreactor would be a logical consideration to facilitate this scale-up as such reactors have been shown to improve nutrient delivery and provide beneficial mechanical forces that support the cultivation of large three-dimensional constructs. It was hypothesized that perfusion culture of tissue-engineered intervertebral disk (IVD) tissues would be capable of generating outer annulus fibrosus (oAF) and nucleus pulposus (NP) tissues comparable with established spinner reactor or static cultures, respectively, without compromising cellular viability, nutrient delivery, and tissue formation. In this study, the perfusion grown oAF and NP tissues did not show a significant difference in extracellular matrix (ECM) quantity or cellular phenotype when compared with their control conditions. In addition, they maintained cellular viability at the center core of the tissues and received enhanced diffusion of medium throughout the tissue when compared with static conditions. This study lays the groundwork for future studies to grow an entire IVD tissue to a physiologically relevant size.

PS1142 DEVELOPMENT OF 3D DRUG‐TESTING PLATFORM FOR CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Conventionally, the efficacy of anti‐cancer drugs in vitro has been tested in the traditional two‐dimensional (2D) cell culture, mostly with plastic platforms. However, in the last few decades, studies have introduced a novel experimental design – a three‐dimensional (3D) cell culture on the so‐called scaffolds. These models have been found to represent a more suitable and accurate approach than the 2D systems (Gurski et al., 2010). Additionally, a co‐culture with other cell types, which are normally present in the tissue, leads to an even closer approximation of the real in vivo situation. Developing an appropriate 3D co‐culture in vitro drug‐testing platform for chronic lymphocytic leukemia (CLL) is of utmost importance, given the microenvironment‐dependency of CLL cells, which normally shortly undergo apoptosis in the conventional 2D cultures, even without any treatment.Aims:To develop an in vitro CLL model which enables a long‐term culture of primary CLL cells and a subsequent drug‐treatment of the cells.Methods:Primary CLL cells were cultured on two types of synthetic materials: i) 0.5% collagen scaffolds and ii) porous hydrogels made of poly(2‐hydroxyl methacrylate‐co‐2‐aminoethyl‐methacrylate) modified with the cell adhesion peptide RGDS [p(HEMA‐co‐AEMA)‐RGDS]. Cell distribution, proliferation, and viability were quantified by AlamarBlue assay or confocal microscopy.Results:We optimized a cell‐seeding method which resulted in a homogenous cell distribution in the pores throughout the whole material. This method required the scaffold to be rotated in the tube with cell suspension with a concentration of 50 million cells per ml. Although overloaded in the first place, the scaffolds later tended to accommodate only hundreds of thousands of cells per piece. The number of living cells on the scaffolds was then maintained by co‐culture with bone marrow stromal cells, which are normally present in the microenvironment of primary CLL cells in vivo. Supplementing the medium with recombinant interleukin 4 (10 ng/μl) and CD40 ligand (1 μg/μl), which were previously found to inhibit apoptosis of CLL cells (Mertens et al., 2012), also contributed to prolonged survival of the cells in vitro. Furthermore, we aimed to support media exchange in the pores of the materials by the gentle motion on a lab rocker. However, this motion was repeatedly proven to be insufficient, since it had no impact on the overall cell survival.Summary/Conclusion:We introduced first steps in creating an appropriate CLL 3D scaffold‐based drug‐testing platform, which mimics the real in vivo CLL microenvironment. The co‐culture on the porous synthetic materials was found to possesses many important advantages, such as larger surface resulting in more frequent cell‐cell and cell‐surface contacts. In the future, we plan to implement either a dynamic growth in a bioreactor with microgravity (Pavesi et al., EHA 2018) or culture in a perfusion reactor (Bourgine et al., 2018).This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the projects CEITEC 2020 (LQ1601), Czech‐Bioimaging large RI project (LM2015062) and MUNI/A/1105/2018; and by the Ministry of Health of the Czech Republic under the research grants AZV‐MZ‐CR 15–31834A and DRO (FNBr, 65269705). The first author is a Brno Ph.D. Talent Scholarship Holder funded by the Brno City Municipality.

Mechanistic role of perfusion culture on bone regeneration

Bone tissue engineering (BTE) aims to develop engineered bone tissue to substitute conventional bone grafts. To achieve this, culturing the cells on the biocompatible three-dimensional (3D) scaffold is one alternative approach. The new functional bone tissue regeneration could be feasible by the synergetic combinations of cells, biomaterials and bioreactors. Although the field of biomaterial design/development for BTE applications attained reasonable success, development of suitable bioreactor remains still a major challenge. Tissue engineering bioreactors provide the microenvironment required for neo-tissue regeneration, and also can be used to study the physio-chemical cues effect on cell proliferation and differentiation in order to produce functional tissue. In this direction, various bioreactors have been developed and evaluated for the successful development of engineered bone tissue. Continues assessment of tissue development and limitations of the bioreactors lead to the progression of perfusion flow bioreactor system. Improvements in perfusion reactor system were able to yield multiple tissue engineered constructs with uniform cell distribution, easy to operate protocols and also effectively handled for the functional tissue development to meet the adequate supply of engineered graft for clinical application.

Larger Pore Size Hollow Fiber Membranes as a Solution to the Product Retention Issue in Filtration-Based Perfusion Bioreactors.

Tangential flow filtration (TFF) and alternating tangential flow (ATF) filtration technologies using hollow fiber membranes are commonly utilized in perfusion cell culture for the production of monoclonal antibodies; however, product retention remains a known and common problem with these systems. To address this issue, commercially available hollow fibers ranging from several hundred kilo-Daltons (kDa) to 0.65 μm in nominal pore size are tested and are all demonstrated to undergo moderate to severe product retention. Further investigation revealed accumulation of particles in the same size range (≈20-200 nm) as the pores. Based on the assumption that these particles contribute to product retention and membrane plugging, a hollow fiber with an unconventionally larger pore size is subsequently identified and demonstrated to drastically reduce product retention with no impact to cell clarification. Furthermore, these hollow fibers demonstrate surprisingly high membrane capacities, making them an attractive solution to the problem of product retention in perfusion reactors.

Improved osteogenic differentiation of umbilical cord blood MSCs using custom made perfusion bioreactor

Background3D cell culture is an appropriate method to develop engineered bone tissue, where different bioreactors have been designed to mitigate the challenges in 3D culture. Currently, we tailored a perfusion reactor to witness human mesenchymal stem cells (MSCs) proliferation and differentiation over polylactic acid-polyethylene glycol (PLA/PEG) composite scaffolds. MethodsThe composite scaffolds with different weight ratios of PLA and PEG were prepared using solvent casting-particulate leaching technique. Human umbilcal card blood MSCs were cultured under dynamic and static conditions to elucidate the role of dynamic fluid flow in osteogenesis of MSCs. ResultsThe human MSCs distribution over the scaffolds was confirmed with fluorescent microscopy. Alkaline phosphatase (ALP), calcium mineralization, and collagen formation were found to be higher in PLA90 scaffolds than PLA100 and PLA75. PLA90 scaffolds with better cell adhesion/proliferartion were considered for bioreactor studies and they exhibited enhanced ALP, Ca+2 mineralization and collagen formation under dynamic perfusion than static culture. We further confirmed our observation by looking at expression levels of osteogenic marker (Runx2 and osteonectin) in differentiated MSCs subjected to perfusion culture compared to static culture. ConclusionThe results of the current investigation once again proves that dynamic perfusion cultures improve the osteogenic differentiation of MSCs over hybrid polymer scaffolds (PLA90) for effective bone regeneration.

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.

Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation

The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA — improvement of compressive strength of calcium phosphate scaffolds – is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts.

Editorial (Thematic Issue: Role of Mesenchymal Stem Cells (MSCs) in Regenerative Medicine and Cell Based Therapies)

Stem cell treatment or therapy is gaining interest as future medicine to treat several dreadful diseases/disorders. Especially mesenchymal stem cells (MSCs) derived from both embryonic and adult cell sources have shown tremendous impact on improving human health. These MSCs have evolved into intensely studied research subjects and clearly collected a great deal of experimental data in the past decade. Even after all these years, research on MSCs still remains a hot and happening area with lot many questions to be answered. In this direction, in our special issue we discussed the safety issue of MSCs as allogenic transplant subject (Sarah Broeckx et al.) and their differentiation potential into cartilage (Vuk Savkovic et al.). We also discussed how nanobiomaterials influence the stem cell modulation (Kiran Kumar Bokara) and how the signaling molecules and signaling pathways influence MSCs fate in regenerative medicine (Rama Raju Baadhe et al. and Birru Bhaskar et al.). In this special issue we also highlighted MSC’s interaction and interference with cancer cells (Chinnapandi Bharathiraja et al.). To the end we discussed advanced bone marrow aspiration by perfusion reactor system.

Effect of Low Culture Temperature on Urokinase Production in Hollow Fiber Reactor

The effect of culture temperature on urokinase production by HT-1080 cell line was studied in batch culture and hollow fiber reactor. Small-scale t-flask experiments revealed that urokinase production could be enhanced and media utilization could be reduced by lowering the culture temperature in production phase. Urokinase production was scaled up using a hollow fiber perfusion reactor system. Temperature of culture was maintained at the physiological 37 °C during growth phase that extended up to 12 days in hollow fiber bioreactor. Subsequently, in the production phase, culture temperature was lowered to 34 °C. Decrease in culture temperature resulted in a significant increase in urokinase production. Proteolytic degradation and inhibition was also minimized. The medium utilization rate was decreased at lower temperature, and hence, a higher economy of production could be obtained.

Online oxygen measurements in ex vivo perfused muscle tissue in a porcine model using dynamic quenching methods

Transplantation of autologous free tissue flaps is the best applicable technique for treating large and complex tissue defects and still has one major failure criterion. Tissue--and in particular muscle tissue--is strongly sensitive to ischemia, thus after a critical period of oxygen depletion the risk of a partial or total flap loss is high. For that reason a miniaturized ex vivo perfusion system has been developed, that supplies the tissue during operational delays. The purpose of this study was to determine the oxygenation levels during such a perfusion using different perfusates and therefore to objectify if a complementary oxygenation unit is required to improve perfusion quality. The oxygen levels of the tissue, as well of the perfusate, were measured by using minimal invasive optical oxygen sensors that are based on dynamic quenching. The ex vivo perfused tissue was the porcine rectus abdominis muscle. Results show, that during perfusion with heparinized crystalloid fluid (Jonosteril) and heparinized autologous whole blood, additional oxygenation of the perfusion reactor led to different ex vivo oxygen tissue saturations, which can be detected by dynamic quenching. Dynamic quenching methods are a promising and valuable technique to perform online oxygen measurements in ex vivo perfused muscle tissue in a porcine model.

A Transient 3D-CFD Model Incorporating Biological Processes for Use in Tissue Engineering

In this article a mathematical model is presented in which the fluid dynamic interaction between the liquid flow in a scaffold and growing cells is simulated. The model is based on a computational fluid dynamic (CFD) model for the representation of the fluid dynamic conditions in the scaffold. It includes furthermore a simple biological growth model based on Michaelis Menten type kinetics for the growth of cells. The model includes biomass, substrate and oxygen as the most important growth limiting components in the system. Furthermore the growth, decay and maintenance respiration of the cells are considered in the model. In a variation of the model the growth of the biomass is influenced by the fluid dynamic induced shear stress level, which the cells are exposed to. In parallel an experimental growth of stem cells has been performed in a 3D perfusion reactor system and the culturing has been stopped after 2, 8 and 13 days. The development of the cells is compared to the simulated growth of cells and it is attempted to draw a conclusion about the impact of the shear stress on the cell growth.

Metabolomics as molecular analysis tool in cell culture engineering

So far, cell culture engineering has used the high-throughput transcriptional and more recently protein profiling techniques attempting to further understand and resolve significant issues regarding the cell culture performance and its optimization. At the metabolic level, flux analysis has been used to monitor changes in the metabolic state that could affect the successful outcome of cell cultures. Metabolomics, referring to the simultaneous quantification of the (relative) concentration of the free small metabolites, is the most recently introduced high-throughput method for the measurement of the metabolic fingerprint of a biological system. Considering the role of metabolism in the context of the overall cellular function, it is easily understandable why quantifying a complete and accurate metabolomic profile is foreseen to have a major positive impact in cell culture engineering research. We will present application of Gas Chromatorgaphy-Mass Spectrometry metabolomics to monitor and analyze mammalian cell culture systems. Specifically, two bioreactor processes will be discussed in which baby hamster kidney cells were cultivated in high-cell density perfusion reactors at both laboratory and manufacturing scales and samples were analyzed over the course of the cultivation. Metabolomics enabled the differentiation between cell cultures based on bioreactor type, and stage of cell growth. Multivariate statistical analysis enabled the identification of the metabolites whose change in concentration was characteristic of the modification in any of these macroscopic parameters. The acquired results support the usefulness of metabolomics as a molecular analysis tool in cell culture engineering.

Metabolomics for high-resolution monitoring of the cellular physiological state in cell culture engineering

Cell culture engineering has to-date used transcriptomic, proteomic, and metabolic flux analyses, attempting to resolve significant questions regarding cell culture performance. Despite the foreseen positive impact, the metabolomic analytical platform has not yet been vastly deployed. Presently, there is no published application of metabolomics to industrial-scale mammalian cell culture. We applied gas chromatography–mass spectrometry metabolomics to analyze baby hamster kidney (BHK) cells of various cell ages cultivated in high-cell density perfusion reactors at both laboratory and manufacturing scales. Cell growth, metabolic activity and protein productivity measurements, which are currently used to monitor the cellular physiological state, suggested consistency across bioreactors and over the course of the cultivation. Nevertheless, metabolomic profiling enabled the differentiation of cell cultures based on cell age, bioreactor scale and cell source. These results support the usefulness of metabolomics as a high resolution molecular analysis tool in cell culture engineering.

Hydrogel‐Perfluorocarbon Composite Scaffold Promotes Oxygen Transport to Immobilized Cells

Cell encapsulation provides cells a three-dimensional structure to mimic physiological conditions and improve cell signaling, proliferation, and tissue organization as compared to monolayer culture. Encapsulation devices often encounter poor mass transport, especially for oxygen, where critical dissolved levels must be met to ensure both cell survival and functionality. To enhance oxygen transport, we utilized perfluorocarbon (PFC) oxygen vectors, specifically perfluorooctyl bromide (PFOB) immobilized in an alginate matrix. Metabolic activity of HepG2 liver cells encapsulated in 1% alginate/10% PFOB composite system was 47-104% higher than alginate systems lacking PFOB. A cubic model was developed to understand the oxygen transport mechanism in the alginate/PFOB composite system. The theoretical flux enhancement in alginate systems containing 10% PFOB was 18% higher than in alginate-only systems. Oxygen uptake rates (OURs) of HepG2 cells were enhanced with 10% PFOB addition under both 20% and 5% O2 boundary conditions, by 8% and 15%, respectively. Model predictions were qualitatively and quantitatively verified with direct experimental OUR measurements using both a perfusion reactor and oxygen sensing plate, demonstrating a greater OUR enhancement under physiological O2 boundary conditions (i.e., 5% O2). Inclusion of PFCs in an encapsulation matrix is a useful strategy for overcoming oxygen limitations and ensuring cell viability and functionality both for large devices (>1 mm) and over extended time periods. Although our results specifically indicate positive enhancements in metabolic activity using the model HepG2 liver system encapsulated in alginate, PFCs could be useful for improving/stabilizing oxygen supply in a wide range of cell types and hydrogels.

Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production

The culture levels of glucose and CO(2) have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO(2) partial pressure (pCO(2)) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 +/- 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h(-1)) as the pCO(2) increased to approximately 220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO(2) up to approximately 220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO(2), both the ratio of lactate to glucose and the alanine production rate were lowered (1.51-1.14 and 17.7-0.56 nmol/10(6) cells h, respectively). Interestingly, when the pCO(2) was increased to approximately 140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at approximately 220 mmHg pCO(2) this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h(-1) and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions.