High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Overcoming challenges in WAVE Bioreactors without feedback controls for pH and dissolved oxygen

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real-time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) transfer--and therefore pH--in the disposable culture chamber. Using a full-factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range--pH 6.8-7.2 and DO exceeding 20% of air saturation--for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred-tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost-effective method for culturing cells in the WAVE Bioreactor.     

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

A hybrid mechanistic-empirical model for in silico mammalian cell bioprocess simulation

In cell culture processes cell growth and metabolism drive changes in the chemical environment of the culture. These environmental changes elicit reactor control actions, cell growth response, and are sensed by cell signaling pathways that influence metabolism. The interplay of these forces shapes the culture dynamics through different stages of cell cultivation and the outcome greatly affects process productivity, product quality, and robustness. Developing a systems model that describes the interactions of those major players in the cell culture system can lead to better process understanding and enhance process robustness. Here we report the construction of a hybrid mechanistic-empirical bioprocess model which integrates a mechanistic metabolic model with subcomponent models for cell growth, signaling regulation, and the bioreactor environment for in silico exploration of process scenarios. Model parameters were optimized by fitting to a dataset of cell culture manufacturing process which exhibits variability in metabolism and productivity. The model fitting process was broken into multiple steps to mitigate the substantial numerical challenges related to the first-principles model components. The optimized model captured the dynamics of metabolism and the variability of the process runs with different kinetic profiles and productivity. The variability of the process was attributed in part to the metabolic state of cell inoculum. The model was then used to identify potential mitigation strategies to reduce process variability by altering the initial process conditions as well as to explore the effect of changing CO₂ removal capacity in different bioreactor scales on process performance. By incorporating a mechanistic model of cell metabolism and appropriately fitting it to a large dataset, the hybrid model can describe the different metabolic phases in culture and the variability in manufacturing runs. This approach of employing a hybrid model has the potential to greatly facilitate process development and reactor scaling.     

A systematic approach for scale-down model development and characterization of commercial cell culture processes

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.     

Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures

With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench‐top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high‐throughput (HT) technology for process development. One such high‐throughput system is the SimCell? platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell? micro‐bioreactor technology for fed‐batch cultivation of a GS‐CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas‐permeable chambers based on a micro‐fluidic design, with six micro‐bioreactors (MBs) per micro‐bioreactor array (MBA). Online, non‐invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3‐ and 100‐L bioreactor scales. The results of the study demonstrate that the SimCell? platform operated under fed‐batch conditions could support viable cell concentrations up to least 12 × 10⁶ cells/mL. In addition, both intra‐MB (MB to MB) as well as intra‐MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra‐MB and ‐MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) × 100. The % CV values for most intra‐MB and intra‐MBA measurements were generally under 10% and the intra‐MBA values were slightly lower than those for intra‐MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench‐top, and pilot scale bioreactor cultivations and found to be within ±20% of the historical averages. Biotechnol. Bioeng. 2010; 106: 57–67. © 2010 Wiley Periodicals, Inc.     

Effects of oxygen and nutrients limitation on ajmalicine production and related enzyme activities in high density cultures of Catharanthus roseus

Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. (c) 1994 John Wiley & Sons, Inc.     

A predictive high‐throughput scale‐down model of monoclonal antibody production in CHO cells

Multi‐factorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of micro‐bioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high‐throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell culture condition. The micro‐bioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 micro‐bioreactors were run for 2 weeks during this DOE experiment to assess this scaled down micro‐bioreactor system as a high‐throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the micro‐bioreactor results with those obtained in conventional 3 L bioreactors. Excellent agreement was observed between the micro‐bioreactor and the bench‐top bioreactor. The micro‐bioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (P < 0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a micro‐scale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods. Biotechnol. Bioeng. 2009; 104: 1107–1120. © 2009 Wiley Periodicals, Inc.     

Correlating on-line monitoring parameters, pH, DO and ORP with nutrient removal in an intermittent cyclic process bioreactor system

The paper presents the study correlating the profile of on-line monitoring parameters and nutrient removal in an intermittent cyclic process bioreactor (ICPBR) system, thereby utilizing the parameters as operational tool. A laboratory scale ICPBR was employed to treat low C/N ratio domestic wastewater from a township. The study was conducted for correlating biological nutrient removal and on-line monitored parameters pH, dissolved oxygen (DO) and oxidation-reduction potential (ORP). The results revealed that pH, DO and ORP related with the dynamic behavior of nutrient concentration (NH4-N, NO3-N, and PO4-P) during treatment in an ICPBR system. The variation in pH and ORP of the reactor liquor correlate to conversion of ammonia (NH4-N) and nitrate (NO3-N) concentrations, respectively. As the bioconversion of ammonia nitrogen and phosphorus are related to the varying profile of the on-line monitored parameters, the profiles could possibly be used as onsite process control parameters.     

Identification of upstream culture conditions and harvest time parameters that affect host cell protein clearance

During early stage bioprocess development, characterizing interactions between unit operations is a key challenge. Such interactions include the release of host cell enzymes early in the process causing losses in product quality downstream. Using a CHO-expressed IgG1 system, the impact of cell culture duration was investigated using a 50 L bioreactor and performing scale-down protein A purification. While antibody titer doubled during the last week of culture, the post-protein A host cell protein (HCP) levels increased from 243 to 740 ppm. Effects of pH and temperature were then explored using fed-batch ambr250 bioreactors, and parameters enabling higher titers were linked to a decrease in post-protein A product purity. These trade-offs between titer and product quality were visualized using a window of operation. The downstream space was explored further by exposing shake flask material to shear representative of disc stack centrifugation, prior to purification, and by adding polishing chromatography. While product quality decreased with progressing cultivation, cells became more shear resistant. Polishing chromatography resulted in product fragmentation which increased fourfold from Day 10 to 24, adding constraint to achieving both efficient HCP clearance as well as high monomer purities. These examples highlight the importance of adopting integrated approaches to upstream and downstream development strategies to enable whole process optimization.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor

The HeLa cell-vaccinia virus expression system was evaluated for the production of recombinant proteins (enhanced green fluorescent protein (EGFP) and HIV envelope coat protein, gp120) using microcarriers in 1.5 L perfused bioreactor cultures. Perfusion was achieved by use of an alternating tangential flow device (ATF), increasing the length of the exponential phase by 50 h compared to batch culture and increasing the maximum cell density from 1.5x10(6) to 4.4x10(6) cell/mL. A seed train expansion method using cells harvested from microcarrier culture and reseeding onto fresh carriers was developed. EGFP was first used as a model protein to study process parameters affecting protein yield, specifically dissolved oxygen (DO) and temperature during the production phase. The highest level of EGFP, 12+/-1.5 microg/10(6) infected cells, was obtained at 50% DO and 31 degrees C. These setpoints were then used to produce glycoprotein, gp120, which was purified and deglycosylated, revealing a significant amount of N-linked glycosylation. Also, biological activity was assayed, resulting in an ID50 of 3.1 microg/mL, which is comparable to previous reports.     

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

Small-scale upstream bioprocess development often occurs in flasks and multi-well plates. These culturing platforms are often not equipped to accurately monitor and control critical process parameters; thus they may not yield conditions representative of manufacturing. In response, we and others have developed optical sensors that enable small-scale process monitoring. Here we have compared two parameters critical to control in industrial cell culture, pH and dissolved oxygen (DO), measured with our optical sensors versus industrially accepted electrochemical probes. For both optical sensors, agreement with the corresponding electrochemical probe was excellent. The Pearson Correlations between the optical sensors and electrochemical probes were 98.7% and 99.7%, for DO and pH, respectively. Also, we have compared optical pH sensor performance in regular (320 mOsm/kg) and high-osmolality (450 mOsm/kg) cell culture media to simulate the increase in osmolality in pH-controlled cultures. Over a pH range of 6.38-7.98 the average difference in pH readings in the two media was 0.04 pH units. In summary, we have demonstrated that these optical sensors agree well with standard electrochemical probes. The accuracy of the optical probes demonstrates their ability to detect potential parameter drift that could have significant impact on growth, production kinetics, and protein product quality. We have also shown that an increase in osmolality that could result from controlling pH or operating the reactor in fed-batch mode has an insignificant impact on the functionality of the pH patches.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Bioreactor controlled by PI algorithm and operated with a perfusion chamber to support endothelial cell survival and proliferation

This paper reports the optimization of a perfusion bioreactor system previously reported by us (Chouinard et al., 2009). The implementation of a proportional-integral (PI) controller algorithm to control oxygen concentration and pH is presented and discussed. P and I values used by the controller were first estimated using a First-Order-Plus-Dead-Time (FOPDT, Matlab Simulink) and then tuned manually. A new gas exchanger design compatible with the PI controller was introduced and validated to decrease interaction between the injected gases and overall inertia of the system. The gas exchanger was used to adjust both pH and dissolved oxygen (DO) concentration. This new bioreactor system allowed real-time PI control over pH and DO concentration at different flow rates (from 2 to 70 mL min(-1)). Cell viability and proliferation were investigated to validate the updated bioreactor design and performance.     

Small-scale bioreactor system for process development and optimization

An agitated 12-well microtiter plate system with a working volume of 2ml was investigated for cell culture process development. Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability. The pH of the NaHCO(3)-buffered system can be adjusted by altering the carbon dioxide content of the gas phase. The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for rapid and precise pH measurements. For high throughputs, cell growth was assessed non-invasively using stable green fluorescent protein (GFP) expressing cells and a fluorescence plate reader. The setup is simple and inexpensive. The system can be automated and allows several hundred small-scale bioreactor experiments to be run in parallel.     

Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.     

生物反应器规模化培养重组CHO-C28细胞表达HBsAg的研究

应用新型聚酯纤维盘片,采用连续灌注培养方式,分别试验了细胞接种量、pH、DO、罐流速度等因素对CHO-C28细胞生长分泌HBsAg的影响,初步建立了5L生物反应器生产重组乙型肝炎疫苗的生产工艺。经3次试验培养,每次培养60d,较适宜的培养条件确定为:pH6.80-7.10,DO 20%-30%,温度36-37℃,灌流速度138ml/h,接种浓度1.9×106cell/ml。收获液的HBsAg平均滴度是1∶256,最高滴度可达1∶512,纯化后的HBsAg产率为0.912mg/L。最后对反应器培养工艺与现行的转瓶培养工艺进行了比较,生物反应器培养具有可控制培养条件、不易污染和可使HBsAg产率提高等优点。     

Real‐time monitoring and control of soluble signaling factors enables enhanced progenitor cell outputs from human cord blood stem cell cultures

Monitoring and control of primary cell cultures is challenging as they are heterogenous and dynamically complex systems. Feedback signaling proteins produced from off‐target cell populations can accumulate, inhibiting the production of the desired cell populations. Although culture strategies have been developed to reduce feedback inhibition, they are typically optimized for a narrow range of process parameters and do not allow for a dynamically regulated response. Here we describe the development of a microbead‐based process control system for the monitoring and control of endogenously produced signaling factors. This system uses quantum dot barcoded microbeads to assay endogenously produced signaling proteins in the culture media, allowing for the dynamic manipulation of protein concentrations. This monitoring system was incorporated into a fed‐batch bioreactor to regulate the accumulation of TGF‐β1 in an umbilical cord blood cell expansion system. By maintaining the concentration of TGF‐β1 below an upper threshold throughout the culture, we demonstrate enhanced ex vivo expansion of hematopoietic progenitor cells at higher input cell densities and over longer culture periods. This study demonstrates the potential of a fully automated and integrated real‐time control strategy in stem cell culture systems, and provides a powerful strategy to achieve highly regulated and intensified in vitro cell manufacturing systems. Biotechnol. Bioeng. 2014;111: 1258–1264. © 2013 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.