Quantitative characterization of virus‐like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

Pease, Leonard F.; Lipin, Daniel I.; Tsai, De-Hao; Zachariah, Michael R.; Lua, Linda H.L.; Tarlov, Michael J.; Middelberg, Anton P. J.

doi:10.1002/bit.22085

Cited by 106 publications

(85 citation statements)

References 46 publications

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“…It should also be pointed out that both equations assume that a ligand, upon adsorption, does not change in size (or conformation); this assumption may not always hold. b) ES-DMA's have been routinely used to characterize particles over a broad size range (~ 3 nm to several hundred nanometers), and has also been validated with several independent liquid and gas phase techniques [14,30,34,75]. Indeed ES-DMA was one of the primary tools in the recent development of NIST traceable nanoparticle size standards [76].…”

Section: Nanoparticle-biomolecule Conjugatesmentioning

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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Section: Nanoparticle-biomolecule Conjugatesmentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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“…A VLP for a simple virus, such as HPV, is a capsid formed by selfassembly of structural protein without the viral genetic material. Recent research in VLP technology is directed towards drug delivery (Forstova et al, 1995;Kimchi-Sarfaty and Gottesman, 2004;Krauzewicz et al, 2000;Lenz et al, 2003) and vaccines (Bright et al, 2008;Galarza et al, 2005;Haynes et al, 2009;Hoffmann et al, 2005;Pushko et al, 2007;Quan et al, 2007;Ross et al, 2009), at times based on chimeric architectures (Gedvilaite et al, 2000;Ionescu et al, 2006;Pease et al, 2009). As with the manufacture of many biotherapeutics (Huang and Leong, 2009), VLP production is susceptible to aggregation.…”

Section: Introductionmentioning

confidence: 98%

Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Ding

Chuan

et al. 2010

Biotech & Bioengineering

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Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self-assembly processing of virus-like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non-native aggregation behavior of self-assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self-assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self-assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second-order concentration dependence, suggesting a dimerization-controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale-up strategies for products that comprise biomolecular assemblies.

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“…The method is based on separation of particles by their ability to resist the flow across the main movement of bulk solution. [4][5][6][7][8][9] In contrast to SEC, large particles have a longer retention time than small ones. The AFFFF technique allows a wide range of molecules, from small proteins to large viruses, to be separated.…”

Section: Introductionmentioning

confidence: 99%

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field‐flow fractionation and multiangle light scattering techniques

Mcevoy¹,

Razinkov²,

Wei³

et al. 2011

Biotechnology Progress

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A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.

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Quantitative characterization of virus‐like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

Cited by 106 publications

References 46 publications

Electrospray–differential mobility analysis of bionanoparticles

Electrospray–differential mobility analysis of bionanoparticles

Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field‐flow fractionation and multiangle light scattering techniques

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