Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application

Kröner, F.; Hubbuch, Jürgen

doi:10.1016/j.chroma.2013.02.017

Cited by 67 publications

(36 citation statements)

References 36 publications

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“…As can be seen in the images, and as quantified by the event analysis in Figure 2E, at salt concentrations higher than 1 mM NaCl, there are fewer active adsorption sites. This result is consistent with ion-exchange elution by salt, as well as ensemble measurements of decreased protein adsorption at increasing salt concentration due to electrostatic shielding [5-15,22,49,50]. …”

Section: Resultssupporting

confidence: 86%

“…Ionic strength and (less frequently) pH often are used to tune adsorption characteristics [22], as they can influence the retention, resolution, and recovery of biomolecules [5-15] and can often provide non-denaturing elution. As discussed below, the observed heterogeneity often is lower at less-adsorptive conditions of higher ionic strength, as inferred from adsorption isotherm fit parameters [12,13].…”

Section: Introductionmentioning

confidence: 99%

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High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

Kisley

Chen

Mansur

et al. 2014

Journal of Chromatography A

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The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

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Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

Kisley

Chen

Mansur

et al. 2014

Journal of Chromatography A

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“…Kröner et al. provided an in silico optimization method of buffer compositions, resulting in well‐controllable pH gradients with low ionic strength validated for characterization of more than 20 proteins .…”

Section: Native Lc Modesmentioning

confidence: 99%

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

Tassi

Vos

Chatterjee

et al. 2017

J of Separation Science

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The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state‐of‐the‐art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid‐chromatographic modes that are discussed include aqueous size‐exclusion chromatography, hydrophobic interaction chromatography, and ion‐exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid‐chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody–drug conjugates is discussed.

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“…Up to this point in the project, for simplicity sake, we had used the buffer system described by Kr€ oner and colleagues 31 for CEX also for our AEX-based purifications by simply switching the start and end buffers (CEX buffer gradient from pH 4.0 to pH 11.0 for CEX resin-based purifications and CEX buffer gradient from pH 11.0 to pH 4.0 for AEX resin-based purifications). However, when we attempted to use the CEX buffer system on the preparative AEX column for the purification of BsAb#6, we noticed the presence of homodimer 1 in all three peaks although the chromatogram had suggested acceptable separation (see chromatogram in Fig.…”

Section: Discovery Of Igg-like Common Light Chain Bsabsmentioning

confidence: 99%

“…31 Their system overcomes the previously described need to compensate gradient non-linearities with a software-enabled, algorithmic control of the gradient mixing, 32 making it simpler and easier to use and implement. Here, we describe the use of this buffer system for the purification of BsAb heterodimers from homodimers on a preparative scale.…”

Section: Introductionmentioning

confidence: 99%

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

Sharkey

Pudi

Moyer

et al. 2016

mAbs

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Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.

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Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application

Cited by 67 publications

References 36 publications

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

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