Probing Submicron Aggregation Kinetics of an IgG Protein by Asymmetrical Flow Field-Flow Fractionation

Bria, Carmen R.M.; Jones, Jeffrey I.; Charlesworth, Alexander; Williams, S. Kim Ratanathanawongs

doi:10.1002/jps.24703

Cited by 12 publications

(6 citation statements)

References 56 publications

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“…Asymmetrical flow-field-flow fractionation (AF4) is an alternative fractionation method to separate aggregates in the submicron range and has potential to monitor aggregation events during early stages 61,79,80 . Dynamic light scattering (DLS) is an in situ technique which is highly sensitive to the formation of aggregates and therefore is very suitable to detect aggregation during early stages, although it cannot resolve size distribution in heterogeneous samples since the signal is strongly biased towards large particles 12 .…”

Section: Experimental Approaches To Directly or Indirectly Characterize Nucleation Events In Biologics Developmentmentioning

confidence: 99%

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

Das

Chou

Jiskoot

et al. 2022

Journal of Pharmaceutical Sciences

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Section: Experimental Approaches To Directly or Indirectly Characterize Nucleation Events In Biologics Developmentmentioning

confidence: 99%

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

Das

Chou

Jiskoot

et al. 2022

Journal of Pharmaceutical Sciences

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“…AF4 provided the essential separation of anti-streptavidin immunoglobulin G (IgG) monomer from aggregates, whereas the MALS-differential refractive index (dRI) reported the MW of the eluting subpopulations. These data were fit to the Lumry-Eyring nucleated polymerization model, and kinetic analysis suggested that slow nucleation and aggregate condensation were the main pathways to the formation of nanometer and submicron aggregates (90). In addition, FFF's characteristic open channel permitted a comparative study of uncentrifuged and centrifuged heat-stressed antistreptavidin IgG.…”

Section: Assemblies and Complexes: Interactions Of Polymers Proteins ...mentioning

confidence: 99%

New Advances and Applications in Field-Flow Fractionation

Plavchak

Smith

Bria

et al. 2021

Annual Rev. Anal. Chem.

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Field-flow fractionation (FFF) is a family of techniques that was created especially for separating and characterizing macromolecules, nanoparticles, and micrometer-sized analytes. It is coming of age as new nanomaterials, polymers, composites, and biohybrids with remarkable properties are introduced and new analytical challenges arise due to synthesis heterogeneities and the motivation to correlate analyte properties with observed performance. Appreciation of the complexity of biological, pharmaceutical, and food systems and the need to monitor multiple components across many size scales have also contributed to FFF's growth. This review highlights recent advances in FFF capabilities, instrumentation, and applications that feature the unique characteristics of different FFF techniques in determining a variety of information, such as averages and distributions in size, composition, shape, architecture, and microstructure and in investigating transformations and function. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 14 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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“…Heat stress has been shown to be an effective method to accelerate the formation of anti-SA IgG1 aggregate species over a wide size range and allow their formation kinetics to be studied. [14,54] and PBS-A2 (high M) sample preparations are shown as the red and the blue shaded areas, respectively. The void peak at 1.5 minutes likely contains large (>~1 µm) material eluting in the steric mode or small fragments of the monomer formed during the heat stress procedure.…”

Section: Preparation Of Low and High Molar Mass Protein Aggregate Sammentioning

confidence: 99%

“…[10] As shown in Figure 1, the AF4 channel has a trapezoidal shape with one wall formed by a semi- where is the channel thickness. [12] In many cases, AF4 also allows greater flexibility in carrier fluid choice compared to SEC [13] and is able to separate over a wide size range (0.01 to >1 µm) with good selectivity (>0.5) [14]. These advantages are at the heart of many publications that provide insights into the formation and dissociation of biological and synthetic complexes and aggregates.…”

Section: Introductionmentioning

confidence: 99%

Impact of asymmetrical flow field-flow fractionation on protein aggregates stability

Bria

Williams

2016

Journal of Chromatography A

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The impact of asymmetrical flow field-flow fractionation (AF4) on protein aggregate species is investigated with the aid of multiangle light scattering (MALS) and dynamic light scattering (DLS). The experimental parameters probed in this study include aggregate stability in different carrier liquids, shear stress (related to sample injection), sample concentration (during AF4 focusing), and sample dilution (during separation). Two anti-streptavidin (anti-SA) IgG1 samples composed of low and high molar mass (M) aggregates are subjected to different AF4 conditions. Aggregates suspended and separated in phosphate buffer are observed to dissociate almost entirely to monomer. However, aggregates in citric acid buffer are partially stable with dissociation to 25% and 5% monomer for the low and high M samples, respectively. These results demonstrate that different carrier liquids change the aggregate stability and low M aggregates can behave differently than their larger counterparts. Increasing the duration of the AF4 focusing step showed no significant changes in the percent monomer, percent aggregates, or the average Ms in either sample. Syringe-induced shear related to sample injection resulted in an increase in hydrodynamic diameter (dh) as measured by batch mode DLS. Finally, calculations showed that dilution during AF4 separation is significantly lower than in size exclusion chromatography with dilution occurring mainly at the AF4 channel outlet and not during the separation. This has important ramifications when analyzing aggregates that rapidly dissociate (<∼2s) upon dilution as the size calculated by AF4 theory may be more accurate than that measured by online DLS. Experimentally, the dhs determined by online DLS generally agreed with AF4 theory except for the more well retained larger aggregates for which DLS showed smaller sizes. These results highlight the importance of using AF4 retention theory to understand the impacts of dilution on analytes.

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Probing Submicron Aggregation Kinetics of an IgG Protein by Asymmetrical Flow Field-Flow Fractionation

Cited by 12 publications

References 56 publications

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

New Advances and Applications in Field-Flow Fractionation

Impact of asymmetrical flow field-flow fractionation on protein aggregates stability

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