Intrinsic fluorescence as an analytical probe of virus-like particle assembly and maturation.
The assembly and maturation of the human papillomavirus (HPV) virus-like particle (VLP) has been monitored by measuring the intrinsic fluorescence intensity using excitation at 290nm and emission at 350nm. The assay was validated to eliminate error due to photo-bleaching, adsorption, and precipitation. Intrinsic fluorescence intensity dropped during both assembly and maturation phases. The decrease during assembly had a second-order dependence on capsomere concentration, as previously observed using light scattering. During post-assembly structural modification the decrease had a first-order dependence on capsomere concentration. Intrinsic fluorescence spectroscopy complements light scattering methodologies for monitoring assembly and enables kinetics of maturation to be observed. The role of environmental factors such as the presence of oxidized glutathione in facilitation of faster and more complete maturation was monitored in real time. Intrinsic fluorescence is a rugged methodology that could be applied to monitoring VLP assembly and maturation unit operations during HPV vaccine manufacturing.
Hanslip
Simon J
SJ
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK.
Zaccai
Nathan R
NR
Middelberg
Anton P J
AP
Falconer
Robert J
RJ
D005453:Fluorescence; D005978:Glutathione / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D052162:Human papillomavirus 16 / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D008027:Light; D046529:Microscopy, Electron, Transmission; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014771:Virion / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D019065:Virus Assembly / Q000187:drug effects
D005978:Glutathione; D019803:Glutathione Disulfide