DNA spike studies for demonstrating improved clearance on chromatographic media

Butler, Michelle D.; Kluck, Brian; Bentley, Tracy

doi:10.1016/j.chroma.2009.08.049

Cited by 19 publications

(18 citation statements)

References 10 publications

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“…The results demonstrated that DNA flows through the column leaving a very low level of DNA in the purified antibody elution fraction with around 2-4 of LRV, running congruently with protein A DNA clearance values. 18,66 …”

Section: Resultsmentioning

confidence: 99%

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

Mustafaoğlu

Kiziltepe

Bilgiçer

2016

Analyst

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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.

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Section: Resultsmentioning

confidence: 99%

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

Mustafaoğlu

Kiziltepe

Bilgiçer

2016

Analyst

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“…The spiking study was divided into two parts: Part 1 was the least favorable conditions for DNA binding (lowest pH and highest conductivity) within the experimental range to determine the minimum DNA clearance capability of the AEX chromatography step. DNA binding in AEX matrices has been shown to be favored at high pH and low conductivity conditions . Combination of low pH and high conductivity would be expected to reduce the electrostatic interactions between DNA and the ligand, by potentially reducing the effective charge of DNA.…”

Section: Resultsmentioning

confidence: 99%

“…DNA binding in AEX matrices has been shown to be favored at high pH and low conductivity conditions. 10,13,14 Combination of low pH and high conductivity would be expected to reduce the electrostatic interactions between DNA and the ligand, by potentially reducing the effective charge of DNA. SuperQ is typically run at a linear velocity ranging from 200 to 500 cm/h.…”

Section: Experimental Design Selectionmentioning

confidence: 99%

Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media

Stone¹,

Borman²,

Ferreira³

et al. 2017

Biotechnology Progress

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Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process‐related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ‐650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141–149, 2018

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“…Enzymatic digestion and selective DNA precipitation may necessitate an additional purification or assay to confirm the complete removal of nuclease and precipitant reagents. AEC was widely applied to reduce hcDNA contamination in monoclonal antibody production [17][18][19], gene therapy vector [12,20,21] and vaccines [7,22,23]. Weak anion exchanger DEAE resin exhibited low adsorption capacity to virus and has been successfully utilized for the production of rabies vaccine [22,23].…”

Section: Introductionmentioning

confidence: 99%

Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium

He¹,

Yang²,

Kuitang³

2011

Journal of Chromatography A

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DNA spike studies for demonstrating improved clearance on chromatographic media

Cited by 19 publications

References 10 publications

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

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

Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media

Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium

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