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Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule.

ABSTRACT: Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the nucleotide-binding site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to multiple advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. Antibody purification was accomplished by first capture of injected samples while running equilibration buffer (50 mM sodium phosphate pH 7.0), followed by elution achieved by running a gradient of mild elution buffer (3 M NaCl in 50 mM phosphate pH 7.0). The results indicate that the m-NBST column efficiency for Rituximab was >98%, with a purity level of >98%. The quality and the capacity of this small molecule membrane affinity purification method is further evaluated for a number of parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen binding activity of antibodies, and column reusability and stability.

SUBMITTER: Mustafaoglu N

PROVIDER: S-EPMC5245175 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule.

Mustafaoglu Nur N Kiziltepe Tanyel T Bilgicer Basar B

The Analyst 20161101 24

Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the nucleotide-binding site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate binding affinity to the NBS. Regenerated cellulose membrane was selected ...[more]

PMID: 27845784

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Project description:BackgroundAffinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system.ResultsElution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml.ConclusionsAffinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

Dataset Information

Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule.

Publications

Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule.

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