Maximizing binding capacity for protein A chromatography

Ghose, Sanchayita; Zhang, Jennifer; Conley, Lynn; Caple, Ryan; Williams, Kevin P.; Cecchini, Douglas J.

doi:10.1002/btpr.1980

Cited by 44 publications

(26 citation statements)

References 20 publications

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“…Unlike its predecessors in the Protein A resin family, the new Protein A ligand on MabSelect SuRe was designed to tolerate alkali solutions by substituting the alkali sensitive amino acid regions with more stable ones . MabSelect SuRe LX has the same functional alkali stable ligand but at much higher density on the bead matrix allowing for higher binding capacities . This permits for the use of more stringent cleaning agents, such as sodium hydroxide, which were once off limits to the Protein A step .…”

Section: Introductionmentioning

confidence: 99%

Maximizing the functional lifetime of Protein A resins

Zhang

Siva

Caple

et al. 2017

Biotechnology Progress

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Protein A chromatography is currently the industry gold-standard for monoclonal antibody and Fc-fusion protein purification. The high cost of Protein A, however, makes resin lifetime and resin reuse an important factor for process economics. Typical resin lifetime studies performed in the industry usually examine the effect of resin re-use on binding capacity, yield, and product quality without answering the fundamental question of what is causing the decrease in performance. A two part mechanistic study was conducted in an attempt to decouple the effect of the two possible factors (resin hydrolysis and/or degradation vs. resin fouling) on column performance over lifetime of the most commonly used alkali-stable Protein A resins (MabSelect SuRe and MabSelect SuRe LX). The change in binding capacity as a function of sodium hydroxide concentration (rate of hydrolysis), temperature, and stabilizing additives was examined. Additionally, resin extraction studies and product cycling studies were conducted to determine cleaning effectiveness (resin fouling) of various cleaning strategies. Sodium hydroxide-based cleaning solutions were shown to be more effective at preventing resin fouling. Conversely, cold temperature and the use of stabilizing additives in conjunction with sodium hydroxide were found to be beneficial in minimizing the rate of Protein A ligand hydrolysis. An effective and robust cleaning strategy is presented here to maximize resin lifetime and thereby the number of column cycles for future manufacturing processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:708-715, 2017.

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Section: Introductionmentioning

confidence: 99%

Maximizing the functional lifetime of Protein A resins

Zhang

Siva

Caple

et al. 2017

Biotechnology Progress

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“…Recent analysis with a 5 g/L bioreactor titer indicated that buffer/product tank volumes would be the primary bottlenecks in the downstream process . A variety of solutions have been evaluated to address these bottlenecks: inline buffer dilution, in‐line product concentration, high‐capacity Protein A, and no‐salt hydrophobic interaction chromatography . These allow for incremental increases in the capacity of the existing purification trains; however, recent improvements in cell culture processes have allowed companies to achieve these higher titers with a shorter duration in the production bioreactor, forcing downstream development groups to explore ways to purify the product even more efficiently.…”

Section: Introductionmentioning

confidence: 99%

Leveraging single‐pass tangential flow filtration to enable decoupling of upstream and downstream monoclonal antibody processing

Brinkmann

Elouafiq

Pieracci

et al. 2018

Biotechnology Progress

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Decoupling upstream and downstream operations in biopharmaceutical production could enable more flexible manufacturing operations and could allow companies to leverage strategic or financial benefits that would be otherwise unattainable. A decoupling process was developed and scaled up utilizing single-pass tangential flow filtration for volume reduction, followed by bulk freezing in single-use bags prior to purification. Single-pass tangential flow filtration can be used to continuously concentrate harvested cell culture fluid, reducing the volume by 15-25× with a step yield of >96%. These concentration factors were reproduced with a second product, indicating that the process could be amenable to platform processes. Experimental data indicate that the product tested was stable for at least one year at -40 or -70°C. The concentration of the harvested cell culture fluid-either with or without a subsequent period of frozen storage-had no impact on the product quality attributes that were tested. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:405-411, 2018.

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“…Also other methods used for the structural determination of protein‐surface interactions require a sample pretreatment resulting in non‐native process conditions as described in the review by Rabe et al. .…”

Section: Introductionmentioning

confidence: 99%

“…We used MabSelect SuRe (GE Healthcare), a popular protein‐A chromatography resin, as the model system and small angle X‐ray scattering (SAXS) as the probing method. Cross‐linked, porous agarose beads 85 μm in diameter are characterized by a rigid, high‐flow backbone matrix and a tetrameric ligand of synthetically engineered B‐domains, called Z‐domain, immobilized via short linkers . From equilibrium binding capacity and ligand density it was derived that on average 3.3 antibody molecules are bound to one MabSelect SuRe protein‐A ligand .…”

Section: Introductionmentioning

confidence: 99%

Antibody adsorption in protein‐A affinity chromatography – in situ measurement of nanoscale structure by small‐angle X‐ray scattering

Plewka

Silva

Tscheließnig

et al. 2018

J of Separation Science

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Protein‐A chromatography is the most widely used chromatography step in downstream processing of antibodies. A deeper understanding of the influence of the surface topology on a molecular/nanoscale level on adsorption is essential for further improvement. It is not clear if the binding is homogenous throughout the entire bead network. We followed the protein absorption process and observed the formation of a protein layer on fibers of chromatography resin in a time‐resolved manner in nanoscale. To characterize the changes in the antibody‐protein‐A ligand complex, small angle X‐ray scattering was employed using a miniaturized X‐ray‐transparent chromatography column packed with a MabSelect SuRe resin. Antibody‐free MabSelect SuRe resin fiber had an average radius of 12 nm and the protein layer thickness resulting from antibody adsorption was 5.5 and 10.4 nm for fiber and junctions, respectively under applied native conditions. We hypothesize that an average of 1.2 antibodies were adsorbed per protein‐A ligand tetramer bound to the outermost units. In contrast to previous studies, it was therefore possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process.

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Maximizing binding capacity for protein A chromatography

Cited by 44 publications

References 20 publications

Maximizing the functional lifetime of Protein A resins

Maximizing the functional lifetime of Protein A resins

Leveraging single‐pass tangential flow filtration to enable decoupling of upstream and downstream monoclonal antibody processing

Antibody adsorption in protein‐A affinity chromatography – in situ measurement of nanoscale structure by small‐angle X‐ray scattering

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