Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.

Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture.

It is highly desirable to prevent crack formation in polymeric materials at an early stage and to extend their lifespan, particularly when repairs to these materials would be difficult for humans. Here, we designed and synthesized catechol-functionalized polymers that can self-heal in seawater through hydrogen bonding and coordination. These bioinspired acrylate polymers are originally viscous materials, but after coordination with environmentally safe, common metal cations in seawater, namely, Ca(2+) and Mg(2+), the mechanical properties of the polymers were greatly enhanced from viscous to tough, hard materials. Reduced swelling in seawater compared with deionized water owing to the higher osmotic pressure resulted in greater toughness (∼5 MPa) and self-healing efficiencies (∼80%).

Current industry practices for large-scale mammalian cell cultures typically employ a standard platform fed-batch process with fixed volume bolus feeding. Although widely used, these processes are unable to respond to actual nutrient consumption demands from the culture, which can result in accumulation of by-products and depletion of certain nutrients. This work demonstrates the application of a fully automated cell culture control, monitoring, and data processing system to achieve significant productivity improvement via dynamic feeding and media optimization. Two distinct feeding algorithms were used to dynamically alter feed rates. The first method is based upon on-line capacitance measurements where cultures were fed based on growth and nutrient consumption rates estimated from integrated capacitance. The second method is based upon automated glucose measurements obtained from the Nova Bioprofile FLEX® autosampler where cultures were fed to maintain a target glucose level which in turn maintained other nutrients based on a stoichiometric ratio. All of the calculations were done automatically through in-house integration with a Delta V process control system. Through both media and feed strategy optimization, a titer increase from the original platform titer of 5 to 6.3 g/L was achieved for cell line A, and a substantial titer increase of 4 to over 9 g/L was achieved for cell line B with comparable product quality. Glucose was found to be the best feed indicator, but not all cell lines benefited from dynamic feeding and optimized feed media was critical to process improvement. Our work demonstrated that dynamic feeding has the ability to automatically adjust feed rates according to culture behavior, and that the advantage can be best realized during early and rapid process development stages where different cell lines or large changes in culture conditions might lead to dramatically different nutrient demands.

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.