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In addition to genomics, transcriptomics and proteomics tools, metabolomics approaches are now used in order to optimize glycoprotein production in CHO cell lines. Recent advances in metabolite quantification have allowed identification of cellular phenotypes under specific experimental conditions (Sellick et al., 2011b). Nutrient utilization and metabolic by-products accumulation are now easily quantifiable and serve as read-outs to improve cell culture conditions. Such tools have particularly contributed to the optimization of feeding cocktails and culture media to increase recombinant glycoprotein production and extend cellular growth (Sellick et al., 2011a, Zang et al., 2011, Chong et al., 2012, Dietmair et al., 2012a, Mohmad-Saberi et al., 2013). Moreover, new targets for cell engineering approaches can be identified, based on metabolomics profiling. A bottleneck at the malate dehydrogenase II (MDHII) level was characterized for the tricarboxylic Deoxycorticosterone acetate synthesis (TCA) cycle in CHO cells and pyruvate metabolism was shown to vary between high producing and low producing anti-CD20 CHO clones (Chong et al., 2010, Ghorbaniaghdam et al., 2014). Finally, a multi-omics study combining transcriptomics and metabolomics data, identified variations in gene expression and in enzymatic reactions during the transition from a parental HEK293 cell line to a producer cell line (Dietmair et al., 2012b).
Conclusions and perspectives
Acknowledgements M.-E. L. was supported by a CIHR (Canadian Institutes of Health Research) fellowship.
Introduction With the rapid development of life science, it is very urgent to develop new technology to analyze and identify glycoproteins quickly and precisely since the biomarkers of many diseases are low-abundance proteins such as glycoproteins, which are low in quantity but significant to organisms [1]. As a result, glycoproteomics has become one of the hottest fields of proteomics nowadays. However, it is a great challenge to enrich low-abundance target proteins and remove high-abundance interfering proteins. Boronate affinity chromatography (BAC) is one of the potential methods for the separation and enrichment of low-abundance glycoproteins, which is based on the specific covalent reaction of boric acid group and cis-diols of glycoproteins [2,3]. The advantage of BAC is that the molecular recognition and selective separation of the glycoproteins is pH-controlled capture/release, and especially suitable for the analysis of trace glycoproteins [4]. However, most of the affinity ligands used in boronate affinity columns were aromatic boronate compounds originally [[5], [6], [7], [8]], which may cause the hydrophobic interaction and produce nonspecific adsorption of proteins or other compounds [9]. In order to overcome this unfavorable interaction, hydrophilic boronic acid ligands and crosslinking monomers have been used to prepare BAC material. For example, 4-vinylphenylboronic acid (VPBA) [10], 3-(acrylamido) phenylboronic acid (AAPBA) [11] and 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) [12] have been used as functional monomers to prepare monolithic boronate affinity material. Furthermore, hydrophilic crosslinking monomers such as pentaerythritol triacrylate (PETA) [13] and N, N’-methylenebisacrylamide (MBAA) [14] were found to reduce the hydrophobic force of BAC significantly. However, the extraction capacity using these strategies was smaller when applied to extraction of glycoproteins in complex samples. In addition, in order to obtain monolith with reliable stability and good permeability, the use of hydrophilic crosslinker required relatively complex porogens in the preparation of BAC. Hence, using hydrophilic co-monomer to prepare BAC may be alternative to solve the problem of nonspecific adsorption of glycoproteins. Oligo (ethylene glycol) methyl ether methacrylate (OEG) is a kind of hydrophilic macromonomer. It contains a short oligo (ethylene glycol) side chain which has been incorporated into polymers to increase the hydrophilicity of polymer materials [15,16]. Besides, the previous work in our group had proved that the incorporation of OEG can increase the hydrophilicity of imprinting cavities and improve the selectivity of molecularly imprinted polymer to template molecules [17]. However, the incorporation of hydrophilic macromonomer into BAC was not studied.