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Anion-Exchange Chromatography

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

Duarte M F Prazeres – 1st expert 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 – 2nd expert 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, Yuan Xu, Gregory S Blank, Kurt Brorson

    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 – 3rd expert 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.