The Experts below are selected from a list of 7401 Experts worldwide ranked by ideXlab platform

Manfred Nimtz - One of the best experts on this subject based on the ideXlab platform.

  • N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster Ovary Cell Line
    FEBS Journal, 2004
    Co-Authors: Guillermina Forno, Mariela Bollati Fogolín, Ricardo Kratje, Harald S. Conradt, Marina Etcheverrigaray, Marcos Oggero, Manfred Nimtz
    Abstract:

    GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different Cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster Ovary Cell Line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1–3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Δm 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal α1–6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found α2–3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.

  • N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster Ovary Cell Line.
    European journal of biochemistry, 2004
    Co-Authors: Guillermina Forno, Mariela Bollati Fogolín, Ricardo Kratje, Harald S. Conradt, Marina Etcheverrigaray, Marcos Oggero, Manfred Nimtz
    Abstract:

    GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different Cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster Ovary Cell Line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Deltam 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal alpha1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found alpha2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.

P D Evans - One of the best experts on this subject based on the ideXlab platform.

  • agonist specific coupling of a cloned drosophila octopamine tyramine receptor to multiple second messenger systems
    The EMBO Journal, 1994
    Co-Authors: S. Robb, John M. Midgley, L.m. Hall, Timothy R Cheek, Frances Hannan, P D Evans
    Abstract:

    A cloned seven transmembrane-spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster Ovary Cell Line, both inhibits adenylate cyclase activity and leads to the elevation of intraCellular Ca2+ levels by separate G-protein-coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy.

  • Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems.
    The EMBO Journal, 1994
    Co-Authors: S. Robb, F.l. Hannan, John M. Midgley, L.m. Hall, Timothy R Cheek, P D Evans
    Abstract:

    A cloned seven transmembrane-spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster Ovary Cell Line, both inhibits adenylate cyclase activity and leads to the elevation of intraCellular Ca2+ levels by separate G-protein-coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy.

Guillermina Forno - One of the best experts on this subject based on the ideXlab platform.

  • N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster Ovary Cell Line
    FEBS Journal, 2004
    Co-Authors: Guillermina Forno, Mariela Bollati Fogolín, Ricardo Kratje, Harald S. Conradt, Marina Etcheverrigaray, Marcos Oggero, Manfred Nimtz
    Abstract:

    GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different Cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster Ovary Cell Line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1–3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Δm 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal α1–6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found α2–3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.

  • N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster Ovary Cell Line.
    European journal of biochemistry, 2004
    Co-Authors: Guillermina Forno, Mariela Bollati Fogolín, Ricardo Kratje, Harald S. Conradt, Marina Etcheverrigaray, Marcos Oggero, Manfred Nimtz
    Abstract:

    GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different Cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster Ovary Cell Line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Deltam 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal alpha1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found alpha2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.

S. Robb - One of the best experts on this subject based on the ideXlab platform.

  • agonist specific coupling of a cloned drosophila octopamine tyramine receptor to multiple second messenger systems
    The EMBO Journal, 1994
    Co-Authors: S. Robb, John M. Midgley, L.m. Hall, Timothy R Cheek, Frances Hannan, P D Evans
    Abstract:

    A cloned seven transmembrane-spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster Ovary Cell Line, both inhibits adenylate cyclase activity and leads to the elevation of intraCellular Ca2+ levels by separate G-protein-coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy.

  • Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems.
    The EMBO Journal, 1994
    Co-Authors: S. Robb, F.l. Hannan, John M. Midgley, L.m. Hall, Timothy R Cheek, P D Evans
    Abstract:

    A cloned seven transmembrane-spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster Ovary Cell Line, both inhibits adenylate cyclase activity and leads to the elevation of intraCellular Ca2+ levels by separate G-protein-coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy.

Alan J. Dickson - One of the best experts on this subject based on the ideXlab platform.

  • determination of chinese hamster Ovary Cell Line stability and recombinant antibody expression during long term culture
    Biotechnology and Bioengineering, 2012
    Co-Authors: Laura Bailey, Ray Field, Diane Hatton, Alan J. Dickson
    Abstract:

    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.

  • determination of chinese hamster Ovary Cell Line stability and recombinant antibody expression during long term culture
    Biotechnology and Bioengineering, 2012
    Co-Authors: Laura Bailey, Ray Field, Diane Hatton, Alan J. Dickson
    Abstract:

    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.