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At‐line multi‐angle light scattering detector for faster process development in enveloped virus‐like particle purification
Author(s) -
Pereira Aguilar Patricia,
GonzálezDomínguez Irene,
Schneider Tobias Amadeus,
Gòdia Francesc,
Cervera Laura,
Jungbauer Alois
Publication year - 2019
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201900441
Subject(s) - dynamic light scattering , fluorescence , light scattering , particle (ecology) , materials science , scattering , chemistry , analytical chemistry (journal) , chromatography , downstream processing , nanoparticle , optics , nanotechnology , physics , biology , ecology
At‐line static light scattering and fluorescence monitoring allows direct in‐process tracking of fluorescent virus‐like particles. We have demonstrated this by coupling at‐line multi‐angle light scattering and fluorescence detectors to the downstream processing of enveloped virus‐like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus‐like particles containing the Human Immunodeficiency Virus‐1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single‐column anion‐exchange chromatography method was used for direct capture and purification. The majority of host‐cell protein impurities passed through the column without binding. Virus‐like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi‐angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus‐like particles were concentrated 17‐fold to final a concentration of 4.45 × 10 10 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.