Biotechnology and Bioengineering

Cover Legend Fully hydrated Staphylococcus epidermidis biofilm imaged by confocal microscopy showing microcolonies (yellow clusters) and extracellular matrix material (red). Each microcolony contains thousands of bacteria. See related article by Stewart et. al., Biotechnology and Bioengineering, Volume 108, Number 12 (December 2011), pages 2968–2977. (Image courtesy of Betsey Pitts, Willy Davison, Paul Perry and Phil Stewart).

This study demonstrates that the unique cold-coacervation properties of resilin-like peptides can be exploited for purification of recombinant proteins co-expressed in E.coli. Purification was achieved without needing affinity chromatography, and with reduced risk of proteolysis that occurs with higher temperature purification systems.

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca²⁺, because binding of Ca²⁺ to Glu residues distal to the active site stabilizes the substrate binding site. Mutations of Glu¹⁰⁵ or Glu¹⁰⁸ in the Ca²⁺-binding site decreased enzyme activity. However, double mutations of Glu¹⁰⁵ and Glu¹⁰⁸ resulted in Ca²⁺-independent activity.

In order to more effectively combat pandemic influenza threats, we need rapid and robust vaccine production methods. Here Welsh and coworkers describe rapid, cost-effective methods for producing the immunoprotective head domain of the influenza surface protein hemagglutinin (HA) using E. coli-based cell-free protein synthesis. After introducing targeted mutations, this protein domain was produced at high yields in either monomeric or trimeric form. The trimer binds sialic acid with the expected affinity suggesting proper folding and full functionality.

Magnetic nanoparticles were immobilized onto Gluconobacter oxydans cells in situ via an adhesive polymer coating inspired by mussel adhesive proteins. G. oxydan aggregates prepared using this method showed high specific activity, ease of separation using a magnet, and good reusability. The approach offers a simple and convenient platform strategy to facilitate magnetization and separation of cells by direct mixing of adhesive nanoparticles with a cell suspension.

Chimeric laccases have been obtained by in vitro and in vivo DNA shuffling of two fungal high redox potential laccases (from Pycnoporus cinnabarinus and PM1 basidiomycetes). The laccase hybrids disclosed in one single round of evolution (using a dual high-throughout screening assay) are active on phenolic and non-phenolic polar substrates and possess combined properties in terms of pH activity and thermostability profiles.

Growth kinetics of microalgae, Chlorella vulgaris, was observed in immobilized arrays of nanoliter-scale microfluidic droplets. The single-cell growth kinetics of C. vulgaris showed significant heterogeneity because the cell-doubling hypothesis does not hold for this microalgae. It was found that the average cell size changes significantly at different stages of cell growth. At the stationary growth phase, the cell size increased with the age of the cell suggesting enhanced accumulation of fatty acids in older cells.

Laboratory-scale bioreactors with an efficient aeration system can sustain yeast culture with high cell density and oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow. Standard models of gas-liquid mass transfer neglect this gradient and fail to predict the observed dynamics and to identify k_La correctly. A new dynamic model together with dimensionless reactor characteristics provides a theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology.

A MALDI TOF-MS method for the quantitative glycomic analysis of CHO cells has been developed. The method allows for the simultaneous analysis of neutral and sialylated glycans with sub-picomolar sensitivity, high accuracy and reproducibility.

This study demonstrates the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. Integration of upstream and downstream unit operations results in the significant simplification of the entire process train due to elimination of non-value-added hold steps, dramatically shorter residence and cycle times, reduction of equipment size, and overall facility minimization.

The paper focuses on the experimental design of a photobioreactor especially conceived for transient isotopic ¹³C-labeling experiments under photoautotrophic conditions. The experimental set-up provided for (i) a rapid sampling and quenching procedure and (ii) rapid mixing throughout the reactor to ensure a sudden step-change in the ¹³C-labeled feed from liquid label injection without affecting uptake flux rate. The operating procedure and performance of the experimental device were tested and validated for continuous cultures of the unicellular green alga Chlamydomonas reinhardtii.

Two aerobic alkane-degrading Pseudomonas can biodefluorinate 8:2, 6:2 and 4:2 fluorotelomer alcohols (FTOHs) into various fluorinated metabolites, including polyfluorinated acids and shorter-chain perfluorinated carboxylic acids (PFCAs). The two Pseudomonas strains biotransformed FTOHs using two different transformation pathways to remove multiple –CF₂–groups from FTOHs to form shorter-chain PFCAs.

Complex protein drugs are addressing unmet medical needs and form a multi-billion-dollar market, but are sometimes difficult to manufacture due to inherent product instability in cell culture and intermediate stages. Vogel and coworkers have developed a new, integrated semi-continuous manufacturing platform using disposable elements, minimizing product residence time and maximizing yield and product quality. Implementation at commercial scale led to a yield improvement of 40%, and robustness has been demonstrated by GMP processing of over 1,000,000L of cell culture harvest.

The present study describes the implementation and evaluation of PTR-MS as a tool for on-line monitoring of volatile organic compounds (VOCs) in the bioreactor headspace. The developed interface between bioreactor and PTR-MS allows for non-invasive, highly reproducible sampling and PTR-MS facilitates online measurement of complex VOC mixtures from ppm down to the ppt level. VOCs are analytes closely related to cell metabolism and the obtained results open promising perspectives in terms of advanced bioprocess monitoring and control.

This study presents a multi-dimensional fractionation and characterization approach for fast acquisition of protein molecular descriptors relevant for chromatographic protein separation from crude biological feedstocks. One application of this approach is that multi-dimensional maps of the obtained molecular properties can be exploited for gaining insight into the complex protein mixture for quick, knowledge-based rational synthesis of purification cascades during downstream process development, as demonstrated by the purification of monoclonal antibody from crude hybridoma cell culture supernatant.

The recovery efficiency diagram can be utilized to define target operating conditions with respect to overconcentration (% OC) and flush volume (N_HUP). It can also be used to assess a system's capability to produce a high concentration formulation outside the original design criteria. The recovery efficiency diagram relates necessary product overconcentration, minimum required product volume at overconcentration, maximum allowable flush volume during product recovery, and the achievable percent product recovery from the system of a given washout kinetic in one diagram.

In this study, the role of autophagy in CHO host cells (DG44) and antibody producing rCHO cells during serum-free suspension culture are investigated using a representative autophagy inducer, rapamycin. The increased autophagy level by rapamycin treatment can delay the apoptosis induction and viability drop, resulting in improved production of antibody-producing rCHO cells. These results imply a positive role of autophagy and the usefulness of pro-autophagy engineering in CHO cell culture.

A metabolomics approach has been used to identify key metabolic differences between high and low antibody-producing CHO cells. Cell samples from eight clones with differing specific productivities (qmAb) derived from the same parent cell line were profiled via liquid chromatography-mass spectrometry. Seven metabolites involved in the key cellular pathways of oxidative phosphorylation, glutathione metabolism and protein glycosylation were shortlisted. This study has resulted in a deeper insight into the metabolic milieu that supports recombinant protein production in mammalian cell cultures.

In the current work, live cell-based sensor cells for detection of cellular responses to TiO₂ NPs were prepared by transfecting an HSP70B' promoter–reporter plasmid. The authors data show the sensor cells have a high response to the TiO₂ NPs exposure. In addition, TiO₂ NPs response shows exposure time and dose dependence manner. Their work should aid in understanding the interactions between bio-nanomaterials and cells.

In the present work, Giobbe and coworkers aimed to study the effect of different confined three-dimensional microenvironments on early differentiation of the three germ layers, in embryoid bodies derived from human embryonic stem cells and human induced pluripotent stem cells. In both cell lines, the local accumulation of soluble secreted factors, given by specific microwell design in a permeable biomaterial, induces defined differentiation patterns.

This work used the global regulator engineering (GRE) approach to obtain mutants of irrE from the radiation-resistant Deinococcus radiodurans which conferred Escherichia coli with markedly enhanced tolerances toward typical inhibitors such as furfural as well as real lignocellulosic hydrolysates. It presents a promising step forward to resolve the hydrolysate inhibitor issue, and suggests the GRE approach can be extended to exploit the exogenous global regulators (e.g., irrE), as well as native ones for eliciting useful phenotypes for industrial microbes.

Immunoelectron micrographs of the c-myc fused RI7/mOR226 chimeric receptors in yeast cells. Immunolabeling was performed with a primary anti-c-myc monoclonal antibody and 10-nm gold-conjugated secondary antibodies. CW, cell wall; CM, cell membrane; Cy, cytoplasm.

A platform-assisted three-dimensional (3D) inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast-based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system.

This work introduces the tissue engineering community to a graphical visualization tool called “Windows of Operation”. This tool allows users to set pre-defined constraint levels for the required performance specification of their system (e.g. cell detachment <20% (blue) or fold change in gene expression >45 (green)) based on different operating conditions (e.g. mean pore size and mean shear stress) and determine whether feasible operating ranges exist that allow the constraints to be met simultaneously (purple).

The suitability of a novel miniaturized hollow fiber bioreactor for pharmacological studies on primary human hepatocytes was investigated. Metabolism of diclofenac, activity and expression of pharmacologically relevant cytochrome P450 enzymes and general metabolism were considerably more stable over the culture period of 10 days compared to conventional monolayer culture. Morevover, bioreactor culture promoted formation of neo-tissue from single cell suspensions with spatial expression patterns resembling native liver tissue.

Nibourg and coworkers used a laboratory version of the AMC-bioartificial liver containing liver cell line HepaRG to test effects of perfusion flow rate through the bioreactor on various hepatic functions and cell damage. The results show that (1) the functionality highly depends on the perfusion rate; (2) there is a universal optimal flow rate; and (3) the mass balance of substrate, metabolite or cell damage markers between in- and out-flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate.

Physiologically similar mechanical strain can revert cultured cells to a more normal phenotype. In this paper Leonard and coworkers show that 3–7% equibiaxial strain results in favorable protein expression in rabbit corneal fibroblasts, and present a novel bioreactor design capable of imparting strains in this range for corneal tissue culture. In addition to more closely resembling the corneal environment, the system is designed such that cultured tissue can be imaged in real time using optical coherence microscopy.

In this communication the authors show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semicrystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium.