The Experts below are selected from a list of 60 Experts worldwide ranked by ideXlab platform

Jürgen Lehmann - One of the best experts on this subject based on the ideXlab platform.

  • Sialidase Activity in Culture Fluid of Chinese Hamster Ovary Cells during Batch Culture and Its Effect on Recombinant Human Antithrombin III Integrity
    Biotechnology progress, 1996
    Co-Authors: Eberhard Munzert, Heino Büntemeyer, Johannes Müthing, Jürgen Lehmann
    Abstract:

    Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human Recombinant Antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal Recombinant protein concentration is highly recommended to avoid product desialylation.

  • Membrane chromatography for rapid purification of Recombinant Antithrombin III and monoclonal antibodies from cell culture supernatant.
    Journal of chromatography, 1993
    Co-Authors: Dirk Lütkemeyer, M. Bretschneider, Heino Büntemeyer, Jürgen Lehmann
    Abstract:

    Abstract The task of purifying monoclonal antibodies (MAbs) and human Recombinant Antithrombin III (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membraneous matrices. The first approach employed a strongly acidic and a strongly basic membrane ion exchanger, which were evaluated for their ability to purify monoclonal antibodies and the human active Recombinant Antithrombin III from cell culture supernatant. Within minutes gram amounts of product could be purified in a high-flux system, specially developed for this purpose, achieving purities of 80% for MAbs and 75% for rATIII, respectively. The capacity of the acidic membrane ion exchanger for MAbs was found to be 1 mg mg/cm 2 with recoveries up to 96% and that of the basic membrane ion exchanger for rATIII was 0.15 mg/cm 2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, for the purification of rATIII. The ATIII binding capacities of the membranes were found to be 91 μg/cm 2 for the amine-modified and 39 μg/cm 2 for the epoxy-activated membrane, achieving purities of 75%. The coupling proved to be fairly stable over a period of 5 months and the membranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances was carried out by gel filtration.

Susan C. Bock - One of the best experts on this subject based on the ideXlab platform.

  • Elimination of glycosylation heterogeneity affecting heparin affinity of Recombinant human Antithrombin III by expression of a β-like variant in baculovirus-infected insect cells
    Biochemical Journal, 1995
    Co-Authors: Eva Ersdal-badju, Xiaoming Peng, Veronique Picard, P. Zendehrouh, Boris Turk, Ingemar Björk, Steven T. Olson, Susan C. Bock
    Abstract:

    In order to promote homogeneity of Recombinant Antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma Antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma Antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma Antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-Antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma Antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of Antithrombin III heparin cofactor activity.

  • Elimination of glycosylation heterogeneity affecting heparin affinity of Recombinant human Antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.
    The Biochemical journal, 1995
    Co-Authors: Eva Ersdal-badju, Xiaoming Peng, Veronique Picard, P. Zendehrouh, Boris Turk, Ingemar Björk, Steven T. Olson, Susan C. Bock
    Abstract:

    In order to promote homogeneity of Recombinant Antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma Antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma Antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma Antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-Antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma Antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of Antithrombin III heparin cofactor activity.

Heino Büntemeyer - One of the best experts on this subject based on the ideXlab platform.

  • Sialidase Activity in Culture Fluid of Chinese Hamster Ovary Cells during Batch Culture and Its Effect on Recombinant Human Antithrombin III Integrity
    Biotechnology progress, 1996
    Co-Authors: Eberhard Munzert, Heino Büntemeyer, Johannes Müthing, Jürgen Lehmann
    Abstract:

    Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human Recombinant Antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal Recombinant protein concentration is highly recommended to avoid product desialylation.

  • Membrane chromatography for rapid purification of Recombinant Antithrombin III and monoclonal antibodies from cell culture supernatant.
    Journal of chromatography, 1993
    Co-Authors: Dirk Lütkemeyer, M. Bretschneider, Heino Büntemeyer, Jürgen Lehmann
    Abstract:

    Abstract The task of purifying monoclonal antibodies (MAbs) and human Recombinant Antithrombin III (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membraneous matrices. The first approach employed a strongly acidic and a strongly basic membrane ion exchanger, which were evaluated for their ability to purify monoclonal antibodies and the human active Recombinant Antithrombin III from cell culture supernatant. Within minutes gram amounts of product could be purified in a high-flux system, specially developed for this purpose, achieving purities of 80% for MAbs and 75% for rATIII, respectively. The capacity of the acidic membrane ion exchanger for MAbs was found to be 1 mg mg/cm 2 with recoveries up to 96% and that of the basic membrane ion exchanger for rATIII was 0.15 mg/cm 2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, for the purification of rATIII. The ATIII binding capacities of the membranes were found to be 91 μg/cm 2 for the amine-modified and 39 μg/cm 2 for the epoxy-activated membrane, achieving purities of 75%. The coupling proved to be fairly stable over a period of 5 months and the membranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances was carried out by gel filtration.

Eva Ersdal-badju - One of the best experts on this subject based on the ideXlab platform.

  • Elimination of glycosylation heterogeneity affecting heparin affinity of Recombinant human Antithrombin III by expression of a β-like variant in baculovirus-infected insect cells
    Biochemical Journal, 1995
    Co-Authors: Eva Ersdal-badju, Xiaoming Peng, Veronique Picard, P. Zendehrouh, Boris Turk, Ingemar Björk, Steven T. Olson, Susan C. Bock
    Abstract:

    In order to promote homogeneity of Recombinant Antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma Antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma Antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma Antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-Antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma Antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of Antithrombin III heparin cofactor activity.

  • Elimination of glycosylation heterogeneity affecting heparin affinity of Recombinant human Antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.
    The Biochemical journal, 1995
    Co-Authors: Eva Ersdal-badju, Xiaoming Peng, Veronique Picard, P. Zendehrouh, Boris Turk, Ingemar Björk, Steven T. Olson, Susan C. Bock
    Abstract:

    In order to promote homogeneity of Recombinant Antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma Antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma Antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma Antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-Antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma Antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of Antithrombin III heparin cofactor activity.

Eberhard Munzert - One of the best experts on this subject based on the ideXlab platform.

  • Sialidase Activity in Culture Fluid of Chinese Hamster Ovary Cells during Batch Culture and Its Effect on Recombinant Human Antithrombin III Integrity
    Biotechnology progress, 1996
    Co-Authors: Eberhard Munzert, Heino Büntemeyer, Johannes Müthing, Jürgen Lehmann
    Abstract:

    Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human Recombinant Antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal Recombinant protein concentration is highly recommended to avoid product desialylation.