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  1. Article ; Online: Use of chelating agents to improve the resolution and consistency of cation-exchange chromatography of monoclonal antibodies.

    Zhang, Liangyi / Robinson, Thomas J / Schmidt, Brian D

    Journal of chromatography. A

    2014  Volume 1367, Page(s) 109–117

    Abstract: Analytical cation-exchange chromatography (CEX) is widely used to profile the charge heterogeneity of therapeutic monoclonal antibodies (mAbs). However, the consistency of CEX profiles of a mAb can be significantly reduced by metal ion impurities from ... ...

    Abstract Analytical cation-exchange chromatography (CEX) is widely used to profile the charge heterogeneity of therapeutic monoclonal antibodies (mAbs). However, the consistency of CEX profiles of a mAb can be significantly reduced by metal ion impurities from sample, mobile phase or leachates from the stainless steel components of the pumping system. In this work, we have developed a new CEX method that dynamically removes metal ions during sample analysis by incorporating the use of chelating agents (1-5mM) in HPLC mobile phases. Among four different chelating agents that were evaluated, EDTA and oxalic acid showed excellent capability of removing metal ions and provided consistent CEX chromatograms for mAb1. Furthermore, the use of oxalic acid in mobile phases not only improved the reproducibility of CEX chromatograms, but also increased the resolution of charge isoforms. Oxalic acid appears capable of binding to mAbs and reducing the positive surface charge density, resulting in a modulation of chromatographic separation. Due to this modulation effect, the CEX resolution was dependent on the concentration of the chelating agent. Optimal resolution for mAb1 was obtained with 2mM of oxalic acid. The oxalic acid modulated CEX method was shown to be capable of monitoring the degradation of mAb1. We further qualified this method according to International Committee on Harmonization (ICH) guidelines and demonstrated that the oxalic acid modulated CEX method is precise and robust at different chromatographic conditions and is suitable for use in a development and/or GMP setting.
    MeSH term(s) Antibodies, Monoclonal/chemistry ; Chelating Agents/chemistry ; Chromatography, High Pressure Liquid/methods ; Chromatography, Ion Exchange/methods ; Limit of Detection ; Reproducibility of Results
    Chemical Substances Antibodies, Monoclonal ; Chelating Agents
    Language English
    Publishing date 2014-11-07
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 1171488-8
    ISSN 1873-3778 ; 0021-9673
    ISSN (online) 1873-3778
    ISSN 0021-9673
    DOI 10.1016/j.chroma.2014.09.051
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 813: Show issues Location:
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  2. Article: Proteolytic DNA for mapping protein-DNA interactions.

    Schmidt, Brian D / Meares, Claude F

    Biochemistry

    2002  Volume 41, Issue 13, Page(s) 4186–4192

    Abstract: We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. ... ...

    Abstract We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. Limited conjugation with the chemical protease FeBABE results in the surface of DNA being randomly labeled at the phosphorothioate sites with this protein-cleaving reagent. After formation of a protein-DNA complex, the proteolytic DNA can be activated to cleave the protein backbone at sites near the DNA. This technique was used to study the bacterial RNA polymerase/lacUV5 DNA open promoter complex, about which significant structural information is available. Cleavage sites on the two largest subunits of RNA polymerase, beta and beta', agree well with a recent model based on the crystal structure of the core enzyme alpha(2)betabeta' [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101, 601-611]. The cleavage site present on alpha supports previous studies regarding DNA binding regions of the alpha subunit. Cleavage sites identified throughout the sigma(70) subunit help to orient it with respect to the open promoter complex.
    MeSH term(s) Bacterial Proteins ; Binding Sites ; DNA/biosynthesis ; DNA/chemistry ; DNA/metabolism ; DNA-Binding Proteins/metabolism ; DNA-Directed RNA Polymerases/genetics ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Genetic Techniques ; Models, Biological ; Models, Molecular ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Protein Binding ; Temperature ; Transcription, Genetic
    Chemical Substances Bacterial Proteins ; DNA-Binding Proteins ; DNA (9007-49-2) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
    Language English
    Publishing date 2002-03-04
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, P.H.S.
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/bi015582r
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 217: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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  3. Article: Principles and methods of affinity cleavage in studying transcription.

    Meares, Claude F / Datwyler, Saul A / Schmidt, Brian D / Owens, Jeffrey / Ishihama, Akira

    Methods in enzymology

    2003  Volume 371, Page(s) 82–106

    MeSH term(s) Binding Sites ; Binding, Competitive ; Biochemistry/methods ; Blotting, Western ; Chelating Agents/pharmacology ; Cross-Linking Reagents/pharmacology ; Cysteine/chemistry ; DNA/chemistry ; DNA/metabolism ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/genetics ; Edetic Acid/analogs & derivatives ; Edetic Acid/chemistry ; Edetic Acid/pharmacology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli/enzymology ; Hydrogen-Ion Concentration ; Ions ; Iron/chemistry ; Models, Chemical ; Models, Molecular ; Mutation ; Peptides/chemistry ; Polymers/chemistry ; Protein Binding ; Thermus/enzymology ; Transcription, Genetic
    Chemical Substances Chelating Agents ; Cross-Linking Reagents ; Ions ; Peptides ; Polymers ; 1-(4-bromoacetamidobenzyl)EDTA (81677-64-7) ; DNA (9007-49-2) ; Edetic Acid (9G34HU7RV0) ; Iron (E1UOL152H7) ; DNA-Directed RNA Polymerases (EC 2.7.7.6) ; Cysteine (K848JZ4886)
    Language English
    Publishing date 2003
    Publishing country United States
    Document type Journal Article
    ISSN 0076-6879
    ISSN 0076-6879
    DOI 10.1016/S0076-6879(03)71006-4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article: Electrophilic chelating agents for irreversible binding of metal chelates to engineered antibodies.

    Chmura, A J / Schmidt, Brian D / Corson, Donald T / Traviglia, Stacey L / Meares, Claude F

    Journal of controlled release : official journal of the Controlled Release Society

    2002  Volume 78, Issue 1-3, Page(s) 249–258

    Abstract: Radiolabeled monoclonal antibodies are widely used in the detection and treatment of cancer. However, several problems still prevent full clinical exploitation of these reagents. Low tumor/background ratios in radioimmunoscintigraphy and high background ... ...

    Abstract Radiolabeled monoclonal antibodies are widely used in the detection and treatment of cancer. However, several problems still prevent full clinical exploitation of these reagents. Low tumor/background ratios in radioimmunoscintigraphy and high background radioactivity in therapy are the foremost among these. The strategy of pretargeting which separates the tumor-targeting step from radiolocalization step may overcome these limitations. One pretargeting approach, based on the streptavidin-biotin system, has been demonstrated to successfully treat cancer in preclinical models (Proc. Natl. Acad. Sci. 97 (2000) 1802). In this report we describe the synthesis of several electrophilic chelates, designed for use in vivo. In this new pretargeting approach, we have used protein engineering to prepare an antibody that can bind selectively and irreversibly to certain of these metal chelates. This improves upon approaches based on the immunogenic protein streptavidin and the endogenous ligand biotin.
    MeSH term(s) Animals ; Antibodies, Monoclonal/metabolism ; Chelating Agents/metabolism ; Drug Stability ; Metals/metabolism ; Mice ; Protein Engineering ; Radioimmunodetection ; Radioimmunotherapy
    Chemical Substances Antibodies, Monoclonal ; Chelating Agents ; Metals
    Language English
    Publishing date 2002-01-28
    Publishing country Netherlands
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, U.S. Gov't, P.H.S.
    ZDB-ID 632533-6
    ISSN 1873-4995 ; 0168-3659
    ISSN (online) 1873-4995
    ISSN 0168-3659
    DOI 10.1016/s0168-3659(01)00485-0
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 2055: Show issues Location:
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