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David C James - One of the best experts on this subject based on the ideXlab platform.
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An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant Monoclonal Antibody Production process titer
Biotechnology and Bioengineering, 2011Co-Authors: Jane Mcleod, Peter M. O'callaghan, Leon P. Pybus, Tracy Root, Stephen J Wilkinson, Andrew J. Racher, David C JamesAbstract:In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) Monoclonal Antibody Production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a Production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab Production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of Production. Instead, cell-specific Mab Production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase Production of a recombinant protein by a mammalian host cell-where control of productivity is inherently protein product and cell line specific. Biotechnol. Bioeng. 2011;108:2193-2204. © 2011 Wiley Periodicals, Inc.
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Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate.
Biotechnology and bioengineering, 2006Co-Authors: Diane M. Dinnis, Andrew J. Racher, Christopher Mark Smales, Scott H. Stansfield, Daniel E. Alete, John R. Birch, Carol T. Marshall, Stefan Schlatter, Lars K Nielsen, David C JamesAbstract:We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant Monoclonal Antibody Production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate Production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein Production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein Production than others and that this bias could be used to select cells that are more likely to achieve high qMab.
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Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate.
Biotechnology and bioengineering, 2004Co-Authors: Christopher Mark Smales, Andrew J. Racher, Diane M. Dinnis, Scott H. Stansfield, Daniel E. Alete, E.a. Sage, John R. Birch, Carol T. Marshall, David C JamesAbstract:We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant Monoclonal Antibody Production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab Production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant Monoclonal Antibody Production.
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functional proteomic analysis of gs ns0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate
Biotechnology and Bioengineering, 2004Co-Authors: Christopher Mark Smales, Andrew J. Racher, Diane M. Dinnis, Scott H. Stansfield, Daniel E. Alete, E.a. Sage, John R. Birch, Carol T. Marshall, David C JamesAbstract:We previously compared changes in individual protein abundance between the proteomes of GS-NSO cell lines with varying rates of cell-specific recombinant Monoclonal Antibody Production (qMab). Here we extend analyses of our proteomic clataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate Production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NSO cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NSO cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NSO cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein Production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein Production than others and that this bias could be used to select cells that are more likely to achieve high qMab. (c) 2006 Wiley Periodicals, Inc.
Andrew J. Racher - One of the best experts on this subject based on the ideXlab platform.
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An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant Monoclonal Antibody Production process titer
Biotechnology and Bioengineering, 2011Co-Authors: Jane Mcleod, Peter M. O'callaghan, Leon P. Pybus, Tracy Root, Stephen J Wilkinson, Andrew J. Racher, David C JamesAbstract:In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) Monoclonal Antibody Production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a Production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab Production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of Production. Instead, cell-specific Mab Production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase Production of a recombinant protein by a mammalian host cell-where control of productivity is inherently protein product and cell line specific. Biotechnol. Bioeng. 2011;108:2193-2204. © 2011 Wiley Periodicals, Inc.
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Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate.
Biotechnology and bioengineering, 2006Co-Authors: Diane M. Dinnis, Andrew J. Racher, Christopher Mark Smales, Scott H. Stansfield, Daniel E. Alete, John R. Birch, Carol T. Marshall, Stefan Schlatter, Lars K Nielsen, David C JamesAbstract:We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant Monoclonal Antibody Production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate Production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein Production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein Production than others and that this bias could be used to select cells that are more likely to achieve high qMab.
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Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate.
Biotechnology and bioengineering, 2004Co-Authors: Christopher Mark Smales, Andrew J. Racher, Diane M. Dinnis, Scott H. Stansfield, Daniel E. Alete, E.a. Sage, John R. Birch, Carol T. Marshall, David C JamesAbstract:We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant Monoclonal Antibody Production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab Production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant Monoclonal Antibody Production.
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functional proteomic analysis of gs ns0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate
Biotechnology and Bioengineering, 2004Co-Authors: Christopher Mark Smales, Andrew J. Racher, Diane M. Dinnis, Scott H. Stansfield, Daniel E. Alete, E.a. Sage, John R. Birch, Carol T. Marshall, David C JamesAbstract:We previously compared changes in individual protein abundance between the proteomes of GS-NSO cell lines with varying rates of cell-specific recombinant Monoclonal Antibody Production (qMab). Here we extend analyses of our proteomic clataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate Production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NSO cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NSO cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NSO cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein Production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein Production than others and that this bias could be used to select cells that are more likely to achieve high qMab. (c) 2006 Wiley Periodicals, Inc.
S. İsmet Gürhan - One of the best experts on this subject based on the ideXlab platform.
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Hybridoma cells immobilized on nonwoven polyester fabric discs: proliferation and Monoclonal Antibody Production in stationary culture.
Journal of biomaterials applications, 2003Co-Authors: Esin Aslankaraoğlu, MenemŞe Gümüşderelioğlu, S. İsmet GürhanAbstract:Mouse hybridoma cells (O146S) producing a Monoclonal Antibody against the 146S antigenic particles of O type apthhoviruses were proliferated on the nonwoven polyester fabric (NWPF) discs in a stationary culture. A series of batch experiments were performed in Petri dishes containing RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum. During the cultivation period glucose and lactate concentrations were measured and Monoclonal Antibody concentration was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The results showed that in the presence of the NWPF discs cell viability, cell yield, and total Monoclonal Antibody Production was higher than that of the disc-free culture.
Christopher Mark Smales - One of the best experts on this subject based on the ideXlab platform.
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Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate.
Biotechnology and bioengineering, 2006Co-Authors: Diane M. Dinnis, Andrew J. Racher, Christopher Mark Smales, Scott H. Stansfield, Daniel E. Alete, John R. Birch, Carol T. Marshall, Stefan Schlatter, Lars K Nielsen, David C JamesAbstract:We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant Monoclonal Antibody Production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate Production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein Production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein Production than others and that this bias could be used to select cells that are more likely to achieve high qMab.
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Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate.
Biotechnology and bioengineering, 2004Co-Authors: Christopher Mark Smales, Andrew J. Racher, Diane M. Dinnis, Scott H. Stansfield, Daniel E. Alete, E.a. Sage, John R. Birch, Carol T. Marshall, David C JamesAbstract:We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant Monoclonal Antibody Production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab Production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant Monoclonal Antibody Production.
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functional proteomic analysis of gs ns0 murine myeloma cell lines with varying recombinant Monoclonal Antibody Production rate
Biotechnology and Bioengineering, 2004Co-Authors: Christopher Mark Smales, Andrew J. Racher, Diane M. Dinnis, Scott H. Stansfield, Daniel E. Alete, E.a. Sage, John R. Birch, Carol T. Marshall, David C JamesAbstract:We previously compared changes in individual protein abundance between the proteomes of GS-NSO cell lines with varying rates of cell-specific recombinant Monoclonal Antibody Production (qMab). Here we extend analyses of our proteomic clataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate Production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NSO cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NSO cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NSO cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein Production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein Production than others and that this bias could be used to select cells that are more likely to achieve high qMab. (c) 2006 Wiley Periodicals, Inc.
Esin Aslankaraoğlu - One of the best experts on this subject based on the ideXlab platform.
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Hybridoma cells immobilized on nonwoven polyester fabric discs: proliferation and Monoclonal Antibody Production in stationary culture.
Journal of biomaterials applications, 2003Co-Authors: Esin Aslankaraoğlu, MenemŞe Gümüşderelioğlu, S. İsmet GürhanAbstract:Mouse hybridoma cells (O146S) producing a Monoclonal Antibody against the 146S antigenic particles of O type apthhoviruses were proliferated on the nonwoven polyester fabric (NWPF) discs in a stationary culture. A series of batch experiments were performed in Petri dishes containing RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum. During the cultivation period glucose and lactate concentrations were measured and Monoclonal Antibody concentration was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The results showed that in the presence of the NWPF discs cell viability, cell yield, and total Monoclonal Antibody Production was higher than that of the disc-free culture.
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Proliferation and Monoclonal Antibody Production of Hybridoma Cells Immobilized on Non-Woven Polyester Fabric Discs
Animal Cell Technology: Basic & Applied Aspects, 2003Co-Authors: Esin Aslankaraoğlu, MenemŞe Gümüşderelioğlu, Ş. Yilmaz, N. BingölAbstract:Mouse hybridoma cells producing Monoclonal Antibody against the 146S antigenic particles of O type apthhoviruses were proliferated on the non-woven polyester fabric (NWPF) discs in stationary and stirred conditions. Firstly, a series of batch experiments were performed in Petri dishes with and without NWPF disc, containing RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum. The results showed that in the presence of the NWPF discs cell viability, cell yield and total Monoclonal Antibody Production was higher than that of the disc-free culture. In the second part, reactor cultures were realized in a 1L spinner basket containing NWPF discs. During the culture the cell growth and MAb Production were examined. The highest MAb concentration were obtained in spinner basket. All results indicated that NWPF-containing spinner basket can be used for the large-scale Production of MAb.