The Experts below are selected from a list of 258 Experts worldwide ranked by ideXlab platform

Duarte M F Prazeres - One of the best experts on this subject based on the ideXlab platform.

  • preparative purification of supercoiled plasmid dna using anion exchange Chromatography
    Journal of Chromatography A, 1998
    Co-Authors: Duarte M F Prazeres, Thomas Schluep, Charles L Cooney
    Abstract:

    Abstract Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-Exchange Chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-Mr RNA, cDNA and plasmid variants. A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-Exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 62±8% yield. Loadings higher than 40 μg/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.

Yuan Xu - One of the best experts on this subject based on the ideXlab platform.

  • generic matrix evaluation of sv40 clearance by anion exchange Chromatography in flow through mode
    Biotechnology and Bioengineering, 2003
    Co-Authors: Sherrie Curtis, Gregory S Blank, Kurt Brorson, Yuan Xu
    Abstract:

    The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) Chromatography to determine if bracketing and generic validation can be applied to anion exchange Chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key Chromatography parameters were identified where an SV40 log10 reduction value (LRV) of ≥4.7 log10 is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange Chromatography, we propose that the concept of “bracketed generic” validation can be applied to this and potentially other Chromatography unit operations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 179–186, 2003.

  • Generic/matrix evaluation of SV40 clearance by anion exchange Chromatography in flow-through mode
    Biotechnology and Bioengineering, 2003
    Co-Authors: Sherrie Curtis, Gregory S Blank, Kurt Brorson, Yuan Xu
    Abstract:

    The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) Chromatography to determine if bracketing and generic validation can be applied to anion exchange Chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key Chromatography parameters were identified where an SV40 log10 reduction value (LRV) of ≥4.7 log10 is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange Chromatography, we propose that the concept of “bracketed generic” validation can be applied to this and potentially other Chromatography unit operations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 179–186, 2003.

Charles L Cooney - One of the best experts on this subject based on the ideXlab platform.

  • preparative purification of supercoiled plasmid dna using anion exchange Chromatography
    Journal of Chromatography A, 1998
    Co-Authors: Duarte M F Prazeres, Thomas Schluep, Charles L Cooney
    Abstract:

    Abstract Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-Exchange Chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-Mr RNA, cDNA and plasmid variants. A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-Exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 62±8% yield. Loadings higher than 40 μg/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.

Karl Mechtler - One of the best experts on this subject based on the ideXlab platform.

  • anion exchange Chromatography of phosphopeptides weak anion exchange versus strong anion exchange and anion exchange Chromatography versus electrostatic repulsion hydrophilic interaction Chromatography
    Analytical Chemistry, 2015
    Co-Authors: Andrew J Alpert, Otto Hudecz, Karl Mechtler
    Abstract:

    Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid Chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion–hydrophilic interaction Chromatography (ERLIC) and Anion-Exchange Chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak Anion-Exchange (WAX) and strong Anion-Exchange (SAX) columns were compared, wit...

Andrew J Alpert - One of the best experts on this subject based on the ideXlab platform.

  • anion exchange Chromatography of phosphopeptides weak anion exchange versus strong anion exchange and anion exchange Chromatography versus electrostatic repulsion hydrophilic interaction Chromatography
    Analytical Chemistry, 2015
    Co-Authors: Andrew J Alpert, Otto Hudecz, Karl Mechtler
    Abstract:

    Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid Chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion–hydrophilic interaction Chromatography (ERLIC) and Anion-Exchange Chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak Anion-Exchange (WAX) and strong Anion-Exchange (SAX) columns were compared, wit...