The Experts below are selected from a list of 32913 Experts worldwide ranked by ideXlab platform
Michael J. Betenbaugh - One of the best experts on this subject based on the ideXlab platform.
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comprehensive glycoproteomic analysis of Chinese Hamster ovary cells
Analytical Chemistry, 2018Co-Authors: Ganglong Yang, Michael J. Betenbaugh, Shisheng Sun, Chuanzi Ouyang, Weiming Yang, Qiong Wang, Hui ZhangAbstract:The Chinese Hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked...
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model based analysis of n glycosylation in Chinese Hamster ovary cells
PLOS ONE, 2017Co-Authors: Frederick J Krambeck, Sandra V Bennun, Mikael Rordam Andersen, Michael J. BetenbaughAbstract:The Chinese Hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese Hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products.
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ultra deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in Chinese Hamster ovary cells
Metabolic Engineering, 2017Co-Authors: Paul S Kelly, Michael J. Betenbaugh, Nicole Borth, Colin Clarke, Alan Costello, Craig Monger, Justine Meiller, Heena Dhiman, Martin Clynes, Niall BarronAbstract:Recent sequencing of the Chinese Hamster ovary (CHO) cell and Chinese Hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese Hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering.
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Chinese Hamster genome database: An online resource for the CHO community at www.CHOgenome.org
Biotechnology and Bioengineering, 2011Co-Authors: Stephanie Hammond, Michael J. Betenbaugh, Mihailo Kaplarevic, Nicole Borth, Kelvin H. LeeAbstract:The Chinese Hamster genome database (http://www.chogenome.org/) is an online resource for the Chinese Hamster (Cricetulus griseus) and Chinese Hamster ovary (CHO) cell communities. CHO cells are important for biomedical research and are widely used in industry for the production of biopharmaceuticals. The genome of the CHO-K1 cell line was recently sequenced and the CHO community has developed an online resource to facilitate accessibility of the genomic data and the development of genomic tools.
Gyun Min Lee - One of the best experts on this subject based on the ideXlab platform.
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a Chinese Hamster transcription start site atlas that enables targeted editing of cho cells
NAR genomics and bioinformatics, 2021Co-Authors: Isaac Shamie, Sascha H. Duttke, K. J. La Cour Karottki, C. Z. Han, Anders Holmgaard Hansen, Hooman Hefzi, Kai Xiong, Jenhan Tao, Samuel J Roth, Gyun Min LeeAbstract:Chinese Hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese Hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese Hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 Hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.
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development of recombinant Chinese Hamster ovary cell lines for therapeutic protein production
Current opinion in chemical engineering, 2013Co-Authors: Soo Min Noh, Madhavi Sathyamurthy, Gyun Min LeeAbstract:The current biopharmaceutical market is driven by the steady increase in demand for recombinant therapeutics produced in mammalian cell lines. Chinese Hamster ovary (CHO) cells are the predominant workhorses for large-scale stable expression of human glycoproteins. However, the low throughput and time-consuming process of cell line development, heterogeneity and instability of CHO cell lines pose serious challenges. This review highlights the conventional expression system with its limitations and the recent advances in high throughput screening methods for rapid clonal selection, targeted gene integration methods for precise prediction of transcriptional activities, and engineering of DNA elements to improve the stability of CHO cell lines.
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development of apoptosis resistant dihydrofolate reductase deficient Chinese Hamster ovary cell line
Biotechnology and Bioengineering, 2003Co-Authors: Suk Lee, Gyun Min LeeAbstract:Chinese Hamster ovary (CHO) cells in serum-batch culture were found to die by apoptosis, which is a physiological and developmental mode of cell death in various types of cells including CHO cells. Apoptotic cell death can be triggered by several adverse conditions such as unavailability of nutrients or serum/growth factors and accumulation of toxic metabolites during cultures. Sodium butyrate (NaBu), which is widely used in rCHO cell cultures for enhanced expression of foreign proteins, can also induce apoptotic cell death of rCHO cells.
Niall Barron - One of the best experts on this subject based on the ideXlab platform.
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ultra deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in Chinese Hamster ovary cells
Metabolic Engineering, 2017Co-Authors: Paul S Kelly, Michael J. Betenbaugh, Nicole Borth, Colin Clarke, Alan Costello, Craig Monger, Justine Meiller, Heena Dhiman, Martin Clynes, Niall BarronAbstract:Recent sequencing of the Chinese Hamster ovary (CHO) cell and Chinese Hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese Hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering.
Keya Chaudhuri - One of the best experts on this subject based on the ideXlab platform.
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activation of mitochondrial promoter ph binding protein in a radio resistant Chinese Hamster cell strain associated with bcl 2
Biochemical and Biophysical Research Communications, 2006Co-Authors: Paromita Roychoudhury, Utpal Ghosh, Nitai P. Bhattacharyya, Keya ChaudhuriAbstract:The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P(H)) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese Hamster V79 cells. These genes are also up regulated in Chinese Hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P(L)) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P(H) promoter in comparison to that observed with cell extracts from Chinese Hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P(H) and mediated by the expression of Bcl2.
Hermann Ragg - One of the best experts on this subject based on the ideXlab platform.
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tyrosine sulfation and n glycosylation of human heparin cofactor ii from plasma and recombinant Chinese Hamster ovary cells and their effects on heparin binding
FEBS Journal, 2002Co-Authors: Christoph Bohme, Harald S. Conradt, Manfred Nimtz, Eckart Grabenhorst, Annemarie Strathmann, Hermann RaggAbstract:The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese Hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with α26 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal α16 fucosylation of oligosacharides from Chinese Hamster ovary cell-derived HCII was detected in > 90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively α23-linked N-acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese Hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin–Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans.