ABSTRACT
A liquid chromatography-mass spectrometry (LC-MS) based metabolomics platform was previously established to identify and profile extracellular metabolites in culture media of mammalian cells. This presented an opportunity to isolate novel apoptosis-inducing metabolites accumulating in the media of antibody-producing Chinese hamster ovary (CHO mAb) fed-batch bioreactor cultures. Media from triplicate cultures were collected daily for the metabolomics analysis. Concurrently, cell pellets were obtained for determination of intracellular caspase activity. Metabolite profiles from the LC-MS data were subsequently examined for their degree of correlation with the caspase activity. A panel of extracellular metabolites, the majority of which were nucleotides/nucleosides and amino acid derivatives, exhibited good (R² > 0.8) and reproducible correlation. Some of these metabolites, such as oxidized glutathione, AMP and GMP, were later shown to induce apoptosis when introduced to fresh CHO mAb cultures. Finally, metabolic engineering targets were proposed to potentially counter the harmful effects of these metabolites.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Recombinant Proteins/chemistry , Adenosine/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis , Bioreactors , CHO Cells , Caspases/metabolism , Cell Cycle , Chromatography, Liquid/methods , Cricetinae , Cricetulus , Mass Spectrometry/methods , MetabolomicsABSTRACT
We have established a liquid chromatography-mass spectrometry based metabolomics platform to identify extracellular metabolites in the medium of recombinant Chinese hamster ovary (CHO) fed-batch reactor cultures. Amongst the extracellular metabolites identified, malate accumulation was the most significant. The contributing factors to malate efflux were found to be the supply of aspartate from the medium, and an enzymatic bottleneck at malate dehydrogenase II (MDH II) in the tricarboxylic acid cycle. Subsequent metabolic engineering to overexpress MDH II in CHO resulted in increases in intracellular ATP and NADH, and up to 1.9-fold improvement in integral viable cell number.
Subject(s)
CHO Cells/cytology , Cell Culture Techniques/methods , Malate Dehydrogenase/biosynthesis , Metabolomics/methods , Animals , Aspartic Acid/metabolism , CHO Cells/metabolism , Cell Count , Cell Growth Processes/physiology , Chromatography, Liquid , Cricetinae , Cricetulus , Malate Dehydrogenase/metabolism , Malates/metabolism , Mass Spectrometry , Metabolic Networks and PathwaysABSTRACT
A metabolomics-based approach was used to time profile extracellular metabolites in duplicate fed-batch bioreactor cultures of recombinant Chinese Hamster Ovary (CHO) cells producing monoclonal IgG antibody. Culture medium was collected and analysed using a high-performance liquid chromatography (HPLC) system in tandem with an LTQ-Orbitrap mass spectrometer. An in-house software was developed to pre-process the LC/MS data in terms of filtering and peak detection. This was followed by principal component analysis (PCA) to assess variance amongst the samples, and hierarchical clustering to categorize mass peaks by their time profiles. Finally, LC/MS2 experiments using the LTQ-Orbitrap (where standard was available) and SYNAPT HDMS (where standard was unavailable) were performed to confirm the identities of the metabolites. Two groups of identified metabolites were of particular interest; the first consisted of metabolites that began to accumulate when the culture entered stationary phase. The majority of them were amino acid derivatives and they were likely to be derived from the amino acids in the feed media. Examples included acetylphenylalanine and dimethylarginine which are known to be detrimental to cell growth. The second group of metabolites showed a downward trend as the culture progressed. Two of them were medium components--tryptophan and choline, and these became depleted midway into the culture despite the addition of feed media. The findings demonstrated the potential of utilizing metabolomics to guide medium design for fed-batch culture to potentially improve cell growth and product titer.
Subject(s)
CHO Cells/metabolism , Metabolome , Metabolomics/methods , Amino Acids/chemistry , Amino Acids/metabolism , Animals , Antibodies, Monoclonal/metabolism , Bioreactors , Cell Culture Techniques/methods , Chromatography, Liquid/methods , Cluster Analysis , Cricetinae , Cricetulus , Culture Media , Dipeptides/chemistry , Dipeptides/metabolism , Immunoglobulin G/metabolism , Mass Spectrometry/methods , Principal Component Analysis , Recombinant Proteins/metabolism , SoftwareABSTRACT
SUMMARY: WEbcoli is a WEb application for in silico designing, analyzing and engineering Escherichia coli metabolism. It is devised and implemented using advanced web technologies, thereby leading to enhanced usability and dynamic web accessibility. As a main feature, the WEbcoli system provides a user-friendly rich web interface, allowing users to virtually design and synthesize mutant strains derived from the genome-scale wild-type E.coli model and to customize pathways of interest through a graph editor. In addition, constraints-based flux analysis can be conducted for quantifying metabolic fluxes and charactering the physiological and metabolic states under various genetic and/or environmental conditions. AVAILABILITY: WEbcoli is freely accessible at http://webcoli.org. CONTACT: cheld@nus.edu.sg.
