Separation and quantification of double‐ and triple‐layered rotavirus‐like particles by CZE

Abstract

Virus-like particles have been successfully used as safe vaccines, as their structure is identical to their native counterparts but devoid of the viral genetic material. However, production of these complex structures is not easy, as recombinant proteins must assemble into virus-like particles. Techniques to differentiate assembled and soluble proteins, as well as assembly intermediaries often present in a sample, are required. An example of complex virus-like particles mixture occurs when rotavirus proteins are recombinantly expressed. Rotavirus-like particles (RLP) can be single (sl), double (dl), or triple layered (tl). The use of RLP preparations as vaccines requires their complete characterization, including separation and quantification of each RLP in a sample. In this work, CZE was evaluated for the separation and quantification of dl and triple-layered rotavirus-like particles (tlRLP). A fused-silica capillary with a deoxycholate running buffer efficiently separated dl and tlRLP in RLP preparations, as they migrated in two discrete peaks with electrophoretic mobilities of 1.24+/-0.04 and 2.95+/-0.03 Ti, respectively. Standard curves for dl and tlRLP were generated, and the response was linearly proportional to analyte concentration. The methodology developed was quantitative, specific, accurate, precise, and reproducible. CZE allowed the quantitative characterization of RLP preparations, which is required for evaluation of immunogens, for process development, and for quality control protocols.

A rapid and simple screening method to identify conditions for enhanced stability of modular vaccine candidates

“…Virus-like particles (VLPs) are macromolecular assemblies of the viral structural protein(s) that retain antigenic features of the authentic virus without the viral genome [1][2][3]. VLPs have come into focus for their promising application in vaccination [4][5][6][7] because of their high safety and efficacy profile [8]; their repetitive and high density native display of epitopes leading to their self-adjuvanting property; their ability to present foreign epitopes on the surface; and their stability compared with soluble antigens [9][10][11][12].…”

Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae

“…RLP were observed by TEM in the bands recovered by ultracentrifugation (Figure 6C and 6D). tlRLP had a morphology similar to that previously reported [25]. Measurements of the RLP obtained were performed using the ImageJ software (Wayne Rasband, NIH, USA), and correspond to the expected size (71 ± 5 nm) for RLP.…”

Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells

“…Both species, tlRLP and dlRLP, were separated in an uncoated fused-silica capillary, using putrescine (1,4-diaminobutane) as dynamic coating agent of the capillary wall. More recently, Castro-Acosta et al [13] improved the separation of tlRLP and dlRLP by CZE, reducing significantly the run time to $20 min and enhancing the peak shape by addition of SDS and deoxycholate to the BGE. Additionally, the methodology was characterized in terms of detection and quantification limits, precision, accuracy, and reproducibility.…”