Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Milhiet, Pierre-Emmanuel; Dosset, Patrice; Godefroy, Cédric; Grimellec, Christian Le; Guigner, Jean‐Michel; Larquet, Éric; Ronzon, Frédéric; Manin, Catherine

doi:10.1016/j.biochi.2010.09.018

Cited by 30 publications

(22 citation statements)

References 22 publications

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“…The TEM sample was dried onto the carbon grid and resulted in the VLP being condensed together, whereas AFM involved a hydrophobic surface. Both methods indicate that most of the VLP is in monomeric form, although no distinct topography can be seen, and the sizes of the particles correlate to that in the literature [34].…”

Section: Effect Of Lipid Removal On Chromatography Processsupporting

confidence: 68%

“…The silanised glass slides were attached to magnetic holders (Agar Scientific, UK) using a protocol from Müller and Engel [33]. The glass slide with the VLP was prepared in a similar method to that stated by Milheit et al [34], with the VLP samples concentrated and diafiltered into Tris-HCl 20 mM, NaCl 150 mM, pH 7.4, using 7 mL Pierce protein concentrators (Thermo Scientific, Loughbough, UK). The VLP was incubated onto the glass slides, for two hours and fixed with 5% glutaraldehyde.…”

Section: Atomic Force Microscopymentioning

confidence: 99%

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A monolith purification process for virus-like particles from yeast homogenate

Burden

Jin

Podgornik

et al. 2012

Journal of Chromatography B

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Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25 mg/mL column.

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Section: Effect Of Lipid Removal On Chromatography Processsupporting

confidence: 68%

Section: Atomic Force Microscopymentioning

confidence: 99%

A monolith purification process for virus-like particles from yeast homogenate

Burden

Jin

Podgornik

et al. 2012

Journal of Chromatography B

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“…Recent developments in non-intrusive biophysical and immunochemical methods that allow analysis of samples in their native state provide the potential for direct measurement and understanding of epitope structure and function [14], [22], [23], [24], [25], [26]. Surface plasmon resonance (SPR) directly and rapidly monitors antigenicity development during epitope refinement in real time without the requirement of a label, where the signal is proportional to the mass deposited onto the sensor chip surface during biomolecular interactions [23].…”

Section: Introductionmentioning

confidence: 99%

“…An optimized solution competitive ELISA, performed with a neutralizing monoclonal antibody (mAb) that targets clinically relevant epitopes [23], quantitatively tracks the development of virion-like epitopes on VLPs in solution or when bound to particulate aluminum-based adjuvant. CryoTEM allows determination of the native, hydrated structure of VLPs [24], [25], and in-solution AFM visualizes VLP surface features without particle deformation or drying [14], [22], [26]. Here, we have evaluated the use of these biophysical and immunochemical methods in concert as a potential novel suite of non-intrusive VLP characterization tools by applying them to the characterization of the VLP-based vaccine against HBV [11].…”

Section: Introductionmentioning

confidence: 99%

Toolbox for Non-Intrusive Structural and Functional Analysis of Recombinant VLP Based Vaccines: A Case Study with Hepatitis B Vaccine

et al. 2012

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BackgroundFundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine.MethodologyThe epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM).Principal FindingsSPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turn-around, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipid-to-protein ratio of HBsAg particles. Results from these non-intrusive biophysical and immunochemical analyses coalesced into a comprehensive understanding of rHBsAg vaccine epitope structure and function that was important for assuring the desired epitope formation, determinants for vaccine potency, and particle stability during vaccine design, development, and manufacturing.SignificanceTogether, the methods presented here comprise a novel suite of non-intrusive VLP structural and functional characterization tools for recombinant vaccines. Key VLP structural features were defined and epitope-specific antigenicity was quantified while preserving epitope integrity and particle morphology. These tools should facilitate the development of other VLP-based vaccines.

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“…[17][18][19] The top of each S-protein tetramer forms a protrusion, where it displays the critical clinically relevant epitopes. 5 The MHR projected out of the lipid layer surface is a tetramer of S-protein monomers, thus containing a total of 32 cysteine residues per protrusion.…”

Section: Discussionmentioning

confidence: 99%