Gill Stephens

Global metabolic profiling of Escherichia coli cultures: An evaluation of methods for quenching and extraction of intracellular metabolites

Metabolomics and systems biology require the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Therefore, we developed and validated standard operating procedures (SOPs) for quenching and efficient extraction of metabolites from Escherichia coli to determine the best methods to approach global analysis of the metabolome. E. coli was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of precisely defined growth conditions, thus enabling sufficient replication of samples. The metabolome profiles were generated using gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). We employed univariate and multivariate statistical analyses to determine the most suitable method. This investigation indicates that 60% cold (-48 degrees C) methanol solution is the most appropriate method to quench metabolism, and we recommend 100% methanol, also at -48 degrees C, with multiple freeze-thaw cycles for the extraction of metabolites. However, complementary extractions would be necessary for coverage of the entire complement of metabolites as detected by GC/TOF-MS. Finally, the observation that metabolite leakage was significant and measurable whichever quenching method is used indicates that methods should be incorporated into the experiment to facilitate the accurate quantification of intracellular metabolites.

Effective Quenching Processes for Physiologically Valid Metabolite Profiling of Suspension Cultured Mammalian Cells

Global metabolite analysis approaches, coupled with sophisticated data analysis and modeling procedures (metabolomics), permit a dynamic read-out of how cellular proteins interact with cellular and environmental conditions to determine cell status. This type of approach has profound potential for understanding, and subsequently manipulating, the regulation of cell function. As part of our study to define the regulatory events that may be used to maximize production of commercially valuable recombinant proteins from cultured mammalian cells, we have optimized the quenching process to allow retention of physiologically relevant intracellular metabolite profiles in samples from recombinant Chinese hamster ovary (CHO) cells. In a comparison of a series of candidate quenching procedures, we have shown that quenching in 60% methanol supplemented with 0.85% ammonium bicarbonate (AMBIC) at -40 degrees C generates a profile of metabolites that is representative of a physiological status based upon examination of key labile cellular metabolites. This represents a key feature for any metabolomic study with suspension cultured mammalian cells and provides confidence in the validity of subsequent data analysis and modeling procedures.

Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Metabolite profiling of industrially important suspension-cultured mammalian cells is being increasingly used for rational improvement of bioprocesses. This requires the generation of global metabolite profiles that cover a broad range of metabolites and that are representative of the cells at the time of sampling. The protocol described here is a validated method for recovery of physiologically relevant amounts of key metabolites from suspension-cultured mammalian cells. The method is a two-step process consisting of initial quenching of the cells (to stop cellular metabolism and allow isolation of the cells) followed by extraction of the metabolites. The cells are quenched in 60% methanol supplemented with 0.85% (wt/vol) ammonium bicarbonate at −40 °C. Metabolites are then extracted from the quenched cells using two 100% methanol extractions followed by a single water extraction. Metabolite samples generated using this protocol are amenable to analysis by mass spectrometry–based techniques (e.g., gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry), NMR spectroscopy and enzymatic assays.

Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets

In this study we report on the optimisation of the technologies for generation of a global metabolomics profile for intracellular metabolites in Chinese hamster ovary (CHO) cells. We evaluated the effectiveness of a range of different extraction methods applied to CHO cells which had been quenched using a previously optimised approach. The extraction methods tested included cold methanol, hot ethanol, acid, alkali and methanol/chloroform plus combinations of these. The extraction of metabolites using two 100% methanol extractions followed by a final water extraction recovered the largest range of metabolites. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery with high reproducibility. Therefore, this was the best extraction method for attaining a global metabolic profile from a single sample. However, another parallel extraction method (e.g. alkali) may also be required to maximise the range of metabolites recovered (e.g. non-polar metabolites).

Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production.

Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the balance of intracellular and extracellular metabolites during the production process of a CHO cell line expressing a recombinant IgG4 antibody. Using this metabolite profiling approach, it was possible to identify nutrient limitations, which acted as bottlenecks for antibody production, and subsequently develop a simple feeding regime to relieve these metabolic bottlenecks. This metabolite profiling-based strategy was used to design a targeted, low cost nutrient feed that increased cell biomass by 35% and doubled the antibody titer. This approach, with the potential for utilization in non-specialized laboratories, can be applied universally to the optimization of production of commercially important biopharmaceuticals.

Highly Enantioselective Reduction of β,β-Disubstituted Aromatic Nitroalkenes Catalyzed by Clostridium sporogenes

This is the first report of the use of Clostridium sporogenes extracts for enantioselective reduction of CC double bonds of beta,beta-disubstituted (1) and alpha,beta-disubstituted nitroalkenes (3). Crude enzyme preparations reduced aryl derivatives 1a-e and 1h, in 35-86% yield with > or =97% ee. Reduction of (E)- and (Z)-isomers of 1c gave the same enantiomer of 2c (> or =99% ee). In contrast, alpha,beta-disubstituted nitroalkene 3a was a poor substrate, yielding (S)- 4a in low yield (10-20%), and the ee (30-70% ee) depended on NADH concentration. An efficient synthesis of a library of nitroalkenes 1 is described.

A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

We have conducted a site‐specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β‐unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2‐phenyl‐1‐nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2‐phenylpropanal oxime from 2‐phenyl‐1‐nitropropene. The multiple binding conformations of hydroxy substituted nitro‐olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.

Catalytic activity of laccases in aqueous solutions of ionic liquids

The ionic liquids, [bmim]Br and [bmim][N(CN)2] (where [bmim] = 1-butyl-3-methylimidazolium), stimulated laccase-catalysed oxidation of catechol when provided at concentrations between 10–20% and 50–60% (v/v) in water, respectively. However, activity was inhibited at higher and lower concentrations. [bmim][BF4] was inhibitory at all concentrations tested, but residual activity was still retained in [bmim][BF4] with ≤ 20% water.

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442.

Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site-Saturated Libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96‐well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site‐saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)‐2‐aryl‐1‐nitropropene and α‐methyl‐trans‐cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β‐unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.