Christopher A. Sellick

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.

Chapter 3 Galactose Metabolism in Yeast-Structure and Regulation of the Leloir Pathway Enzymes and the Genes Encoding Them

The enzymes of the Leloir pathway catalyze the conversion of galactose to a more metabolically useful version, glucose-6-phosphate. This pathway is required as galactose itself cannot be used for glycolysis directly. In most organisms, including the yeast Saccharomyces cerevisiae, five enzymes are required to catalyze this conversion: a galactose mutarotase, a galactokinase, a galactose-1-phosphate uridyltransferase, a UDP-galactose-4-epimerase, and a phosphoglucomutase. In yeast, the genes encoding these enzymes are tightly controlled at the level of transcription and are only transcribed under specific sets of conditions. In the presence of glucose, the genes encoding the Leloir pathway enzymes (often called the GAL genes) are repressed through the action of a transcriptional repressor Mig1p. In the presence of galactose, but in the absence of glucose, the concerted actions of three other proteins Gal4p, Gal80p, and Gal3p, and two small molecules (galactose and ATP) enable the rapid and high-level activation of the GAL genes. The precise molecular mechanism of the GAL genetic switch is controversial. Recent work on solving the three-dimensional structures of the various GAL enzymes proteins and the GAL transcriptional switch proteins affords a unique opportunity to delve into the precise, and potentially unambiguous, molecular mechanism of a highly exploited transcriptional circuit. Understanding the details of the transcriptional and metabolic events that occur in this pathway can be used as a paradigm for understanding the integration of metabolism and transcriptional control more generally, and will assist our understanding of fundamental biochemical processes and how these might be exploited.

Molecular structure of Saccharomyces cerevisiae Gal1p, a bifunctional galactokinase and transcriptional inducer.

Gal1p of Saccharomyces cerevisiae is capable of performing two independent cellular functions. First, it is a key enzyme in the Leloir pathway for galactose metabolism where it catalyzes the conversion of α-d-galactose to galactose 1-phosphate. Second, it has the capacity to induce the transcription of the yeast GAL genes in response to the organism being challenged with galactose as the sole source of carbon. This latter function is normally performed by a highly related protein, Gal3p, but in its absence Gal1p can induce transcription, albeit inefficiently, both in vivo and in vitro. Here we report the x-ray structure of Gal1p in complex with α-d-galactose and Mg-adenosine 5′-(β,γ-imido)triphosphate (AMPPNP) determined to 2.4 Å resolution. Overall, the enzyme displays a marked bilobal appearance with the active site being wedged between distinct N- and C-terminal domains. Despite being considerably larger than other galactokinases, Gal1p shares a similar molecular architecture with these enzymes as well as with other members of the GHMP superfamily. The extraordinary levels of similarity between Gal1p and Gal3p (∼70% amino acid identity and ∼90% similarity) have allowed a model for Gal3p to be constructed. By identifying the locations of mutations of Gal3p that result in altered transcriptional properties, we suggest potential models for Gal3p function and mechanisms for its interaction with the transcriptional inhibitor Gal80p. The GAL genetic switch has long been regarded as a paradigm for the control of gene expression in eukaryotes. Understanding the manner in which two of the proteins that function in transcriptional regulation interact with one another is an important step in determining the overall molecular mechanism of this switch.

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.

Modulation of transcription factor function by an amino acid: activation of Put3p by proline

Saccharomyces cerevisiae are able to convert proline to glutamate so that it may be used as a source of nitrogen. Here, we show that the activator of the proline utilization genes, Put3p, is transcriptionally inert in the absence of proline but transcriptionally active in its presence. The activation of Put3p requires no additional yeast proteins and can occur in the presence of certain proline analogues: an unmodified pyrrolidine ring is able to activate Put3p as efficiently as proline itself. In addition, we show that a direct interaction occurs between Put3p and proline. These data, which represent direct control of transcriptional activator function by a metabolite, are discussed in terms of the regulation of proline‐specific genes in yeast and as a general mechanism of the control of transcription.

The challenge of applying Raman spectroscopy to monitor recombinant antibody production

UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.

Understanding a transcriptional paradigm at the molecular level : the structure of yeast gal80p

In yeast, the GAL genes encode the enzymes required for normal galactose metabolism. Regulation of these genes in response to the organism being challenged with galactose has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins, the activator Gal4p, the repressor Gal80p, and the ligand sensor Gal3p, control the switch between inert and active gene expression. Gal80p, the focus of this investigation, plays a pivotal role both in terms of repressing the activity of Gal4p and allowing the GAL switch to respond to galactose. Here we present the three-dimensional structure of Gal80p from Kluyveromyces lactis and show that it is structurally homologous to glucose-fructose oxidoreductase, an enzyme in the sorbitol-gluconate pathway. Our results clearly define the overall tertiary and quaternary structure of Gal80p and suggest that Gal4p and Gal3p bind to Gal80p at distinct but overlapping sites. In addition to providing a molecular basis for previous biochemical and genetic studies, our structure demonstrates that much of the enzymatic scaffold of the oxidoreductase has been maintained in Gal80p, but it is utilized in a very different manner to facilitate transcriptional regulation.