Robustness of virus removal by protein A chromatography is independent of media lifetime

Lute, Scott; Norling, Lenore A.; Hanson, Michael A.; Emery, Rachel; Stinson, Denise; Padua, Kevin; Blank, G.; Chen, Qi; Brorson, Kurt

doi:10.1016/j.chroma.2008.07.094

Cited by 47 publications

(40 citation statements)

References 36 publications

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“…For both types of viruses, load density and pooling does not impact LRVs substantially. Historically, somewhat higher LRVs are obtained with end-of-use than naïve resin (Brorson et al, 2003a;Chen, unpublished observations;Lute et al, 2008). The same trend occurred in this study for both viruses (two of two experiments).…”

Section: Resultssupporting

confidence: 83%

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Zhang

Lute

Norling

et al. 2008

Biotech & Bioengineering

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Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

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Section: Resultssupporting

confidence: 83%

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Zhang

Lute

Norling

et al. 2008

Biotech & Bioengineering

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“…A key issue in the bioprocessing of IgG using protein A affinity chromatography columns is the ability to purify multiple cycles of cell lysate on the same bed, ensuring successful regeneration without loss of capacity and no leakage of protein A from the support . While an extensive cycling lifetime study is certainly warranted, it is informative to evaluate the rSPA‐modified surface under solvent conditions common in HPLC and SPE methods as well as common IgG elution buffers.…”

Section: Resultsmentioning

confidence: 99%

Initial evaluation of protein A modified capillary-channeled polymer fibers for the capture and recovery of immunoglobulin G

Schadock-Hewitt

Marcus

2014

J. Sep. Science

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A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary-channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary-channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.

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“…Due to the specific adsorption of antibodies, it is widely used for antibody capture from harvest and removal of process-related impurities including HCP, DNA and viruses [1][2][3]. However, it is rarely used for removal of an important class of product-related impurities, antibody aggregates from harvest, due species.…”

Section: Introductionmentioning

confidence: 99%

Molecular perspective of antibody aggregates and their adsorption on Protein A resin

Song

Huang

et al. 2016

Journal of Chromatography A

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Robustness of virus removal by protein A chromatography is independent of media lifetime

Cited by 47 publications

References 36 publications

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Initial evaluation of protein A modified capillary-channeled polymer fibers for the capture and recovery of immunoglobulin G

Molecular perspective of antibody aggregates and their adsorption on Protein A resin

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