The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals

Arakawa, Tsutomu; Ejima, Daisuke; Li, Tiansheng; Philo, John S.

doi:10.1002/jps.21974

Cited by 198 publications

(121 citation statements)

References 106 publications

(141 reference statements)

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“…Dilution is a limitation of this method since it can inuence the material's content in relative percentage of each species, as it can be a factor for some aggregates to disassociate, and therefore be considered reversible. 37,38 It was important to understand if this was the case with rAlbumin solutions. By comparing injections of proteins at 50 mg mL À1 and 10 mg mL À1 concentrations, their respective peak areas were different by factors of <1% (see Fig.…”

Section: High-performance Size Exclusion Chromatography (Hpsec)mentioning

confidence: 99%

The effect of protein concentration on the viscosity of a recombinant albumin solution formulation

et al. 2016

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ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. The effect of protein concentration on solution viscosity in a commercially available biopharmaceutical formulation of recombinant albumin (rAlbumin) was studied. The level of protein aggregation with concentration and its impact on solution viscosity was investigated. Theoretical models predicting viscosity with concentration were applied to these data, and a model that accounts for multiple protein species in solution provided the best fit. The results highlight the need to account for heterogeneity in the level of aggregation when addressing the increase of viscosity observed at high concentrations of protein solutions, a significant issue for the manufacture and use of protein-based therapeutics.

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Section: High-performance Size Exclusion Chromatography (Hpsec)mentioning

confidence: 99%

The effect of protein concentration on the viscosity of a recombinant albumin solution formulation

et al. 2016

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“…Secondly, high salt concentrations in the mobile phase of the SEC are used to minimize adsorption of protein, but it is known that high salt concentrations can also break up aggregates [5]. Thirdly, prior work by others has indicated that dimers may preferentially absorb in the SEC column [200,204]. Inaccuracies can also arise in determining the dimer/monomer ratio using the ES-DMA.…”

Section: Comparison Of Es-dma With Secmentioning

confidence: 99%

“…Because protein aggregates span a wide range of sizes, and quantification of aggregates depends on the technique employed, there is no one analytical tool that can measure all classes of aggregates [3,199,200], and multiple methods are typically used for cross validation [5,197,199]. The objective of this paper is to evaluate electrospraydifferential mobility analysis (ES-DMA) as another method to add to the suite of protein aggregate characterization tools.…”

Section: Introductionmentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

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“…Non-specific interactions between antibodies and SEC resins under aqueous conditions are well-documented and believed to be dominated by electrostatic interactions. 24 This phenomenon is typically abrogated with high ionic strength mobile phase, an approach not well-suited for subsequent isoelectric focusing. 25 To alter any potential electrostatic interactions, the chromatography was performed in mildly acidic mobile phase (pH 4.5) so that both the heavy and light chains would have a net positive charge.…”

Section: Resultsmentioning

confidence: 99%

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

Vanam

Schneider

Marlow

2015

mAbs

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To cite this article: Ram P Vanam, Michael A Schneider & Michael S Marlow (2015) Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity, mAbs, 7:6, 1118-1127, DOI: 10.1080DOI: 10. /19420862.2015 An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

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The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals

Cited by 198 publications

References 106 publications

The effect of protein concentration on the viscosity of a recombinant albumin solution formulation

The effect of protein concentration on the viscosity of a recombinant albumin solution formulation

Electrospray–differential mobility analysis of bionanoparticles

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

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