The Experts below are selected from a list of 43053 Experts worldwide ranked by ideXlab platform
Subinay Ganguly - One of the best experts on this subject based on the ideXlab platform.
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early prediction of instability of chinese hamster Ovary Cell lines expressing recombinant antibodies and antibody fusion proteins
Biotechnology and Bioengineering, 2012Co-Authors: Haimanti Dorai, Susanne Corisdeo, Dawn Ellis, Cherylann Kinney, Matt Chomo, Pam Hawleynelson, Gordon Moore, Michael J Betenbaugh, Subinay GangulyAbstract:One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian Cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster Ovary (CHO) derived production Cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a Cell line expressing an antibody or antibody-fusion protein declined by 20–30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production Cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of Cells as detected with flow cytometric analysis of intraCellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host Cell line from which the candidate production Cell lines were derived was apoptotic-resistant. This data suggested that unstable Cell lines were more prone to apoptosis, which was confirmed by the fact that unstable Cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production Cell lines at an early stage of the Cell line development process, potentially reducing the cost of biotherapeutic development. Biotechnol. Bioeng. 2012; 109:1016–1030. © 2011 Wiley Periodicals, Inc.
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early prediction of instability of chinese hamster Ovary Cell lines expressing recombinant antibodies and antibody fusion proteins
Biotechnology and Bioengineering, 2012Co-Authors: Haimanti Dorai, Susanne Corisdeo, Dawn Ellis, Cherylann Kinney, Matt Chomo, Pam Hawleynelson, Gordon Moore, Michael J Betenbaugh, Subinay GangulyAbstract:One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian Cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster Ovary (CHO) derived production Cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a Cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production Cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of Cells as detected with flow cytometric analysis of intraCellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host Cell line from which the candidate production Cell lines were derived was apoptotic-resistant. This data suggested that unstable Cell lines were more prone to apoptosis, which was confirmed by the fact that unstable Cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production Cell lines at an early stage of the Cell line development process, potentially reducing the cost of biotherapeutic development.
Alan J. Dickson - One of the best experts on this subject based on the ideXlab platform.
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determination of chinese hamster Ovary Cell line stability and recombinant antibody expression during long term culture
Biotechnology and Bioengineering, 2012Co-Authors: Laura Bailey, Ray Field, Diane Hatton, Alan J. DicksonAbstract:Chinese hamster Ovary (CHO) Cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO Cells for therapeutic protein production is the selection of Cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO Cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this Cell line, stability of expression was retained for 40–50 generations after which time a 40% loss in antibody production was detected. The instability observed within the Cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative Cell time values and an apparent increased sensitivity to Cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in Cellular metabolism during LTC (alterations to extraCellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO Cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning Cellular phenotype changes require further detailed analysis at a molecular level. Biotechnol. Bioeng. 2012; 109:2093–2103. © 2012 Wiley Periodicals, Inc.
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determination of chinese hamster Ovary Cell line stability and recombinant antibody expression during long term culture
Biotechnology and Bioengineering, 2012Co-Authors: Laura Bailey, Ray Field, Diane Hatton, Alan J. DicksonAbstract:Chinese hamster Ovary (CHO) Cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO Cells for therapeutic protein production is the selection of Cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO Cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this Cell line, stability of expression was retained for 40-50 generations after which time a 40% loss in antibody production was detected. The instability observed within the Cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative Cell time values and an apparent increased sensitivity to Cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in Cellular metabolism during LTC (alterations to extraCellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO Cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning Cellular phenotype changes require further detailed analysis at a molecular level.
Gyun Min Lee - One of the best experts on this subject based on the ideXlab platform.
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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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selection strategies for the establishment of recombinant chinese hamster Ovary Cell line with dihydrofolate reductase mediated gene amplification
Applied Microbiology and Biotechnology, 2005Co-Authors: Seung Chul Jun, Min Soo Kim, Jong Youn Baik, Sun Ok Hwang, Gyun Min LeeAbstract:To evaluate the efficacy of selection strategies for recombinant Chinese hamster Ovary (rCHO) clones undergone with dihydrofolate reductase-mediated gene amplification, rCHO Cell lines producing a chimeric antibody were established using two strategies, one based on individual clones and the other based on Cell pools. In a selection based on individual clones, Cell cloning by limiting dilution method was performed twice, once after a round of selection of parental Cell clones and once after obtaining high-producer clones. Thirty parental clones selected from 300 parental clones were cultivated independently throughout the gene amplification procedure. Using this labor-intensive strategy, it took approximately 17 weeks to obtain high-producing clones such as CS11-8 and CS18-3 clones. A selection based on Cell pools, in which Cell cloning was performed once at the final selection stage, required less effort and time to amplify large numbers of individual parental clones within the pool. However, high-producing clones were lost during the amplification procedure. The antibody expression level of high-producing clones such as PS7-2 and PS7-32 chosen on the basis of Cell pools was less than one third of that of CS11-8 and CS18-3 clones. Taken together, a selection strategy based on individual clones is favored for establishment of high-producing rCHO clones because it is more efficient to perform Cell cloning at the initial selection stage of parental Cell clones.
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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.
Bahram Kazemi - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression of soluble vascular endothelial growth factors receptor 1 sflt 1 fragments in cho k1
International Journal of Clinical and Experimental Medicine, 2013Co-Authors: Poopak Farnia, Jalaledin Ghanavi, Mojgan Bandehpour, Bahram KazemiAbstract:Soluble form of vascular endothelial growth factor (VEGF) receptor (sFlt-1) has been considered a key target in anti angiogenesis strategies. In this study, sFlt-1 was amplified and cloned. For recombinant production of sFlt-1 protein, Chinese hamster Ovary Cell line (CHO) was used. The liposome–mediated transfection with sFlt-1 gene, were detected in sFlt-1 positive Cells as early as 24 hours post-transfection. The production of sFlt-1 protein was confirmed using SDS-PAGE and immune blotting results. In present investigation, the recombinant protein of sFlt-1 had expressed with correct folding. The system is economically applicable for large production of sFlt protein and can be used as further therapeutic approaches in targeting the growth of solid tumor tissue.
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cloning and expression of soluble vascular endothelial growth factors receptor 1 sflt 1 fragments in cho k1
International Journal of Clinical and Experimental Medicine, 2013Co-Authors: Poopak Farnia, Jalaledin Ghanavi, Mojgan Bandehpour, Bahram KazemiAbstract:Soluble form of vascular endothelial growth factor (VEGF) receptor (sFlt-1) has been considered a key target in anti angiogenesis strategies. In this study, sFlt-1 was amplified and cloned. For recombinant production of sFlt-1 protein, Chinese hamster Ovary Cell line (CHO) was used. The liposome–mediated transfection with sFlt-1 gene, were detected in sFlt-1 positive Cells as early as 24 hours post-transfection. The production of sFlt-1 protein was confirmed using SDS-PAGE and immune blotting results. In present investigation, the recombinant protein of sFlt-1 had expressed with correct folding. The system is economically applicable for large production of sFlt protein and can be used as further therapeutic approaches in targeting the growth of solid tumor tissue.
Haimanti Dorai - One of the best experts on this subject based on the ideXlab platform.
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early prediction of instability of chinese hamster Ovary Cell lines expressing recombinant antibodies and antibody fusion proteins
Biotechnology and Bioengineering, 2012Co-Authors: Haimanti Dorai, Susanne Corisdeo, Dawn Ellis, Cherylann Kinney, Matt Chomo, Pam Hawleynelson, Gordon Moore, Michael J Betenbaugh, Subinay GangulyAbstract:One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian Cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster Ovary (CHO) derived production Cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a Cell line expressing an antibody or antibody-fusion protein declined by 20–30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production Cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of Cells as detected with flow cytometric analysis of intraCellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host Cell line from which the candidate production Cell lines were derived was apoptotic-resistant. This data suggested that unstable Cell lines were more prone to apoptosis, which was confirmed by the fact that unstable Cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production Cell lines at an early stage of the Cell line development process, potentially reducing the cost of biotherapeutic development. Biotechnol. Bioeng. 2012; 109:1016–1030. © 2011 Wiley Periodicals, Inc.
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early prediction of instability of chinese hamster Ovary Cell lines expressing recombinant antibodies and antibody fusion proteins
Biotechnology and Bioengineering, 2012Co-Authors: Haimanti Dorai, Susanne Corisdeo, Dawn Ellis, Cherylann Kinney, Matt Chomo, Pam Hawleynelson, Gordon Moore, Michael J Betenbaugh, Subinay GangulyAbstract:One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian Cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster Ovary (CHO) derived production Cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a Cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production Cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of Cells as detected with flow cytometric analysis of intraCellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host Cell line from which the candidate production Cell lines were derived was apoptotic-resistant. This data suggested that unstable Cell lines were more prone to apoptosis, which was confirmed by the fact that unstable Cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production Cell lines at an early stage of the Cell line development process, potentially reducing the cost of biotherapeutic development.
